Vol.3 No.1
2023 December
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OPERATIVE DENTISTRY, ENDODONTOLOGY AND PERIODONTOLOGY
Vol. 3, No. 1

CONTENTSDECEMBER 2023

Original Articles
Effect of Apical Limit Settings on the Occurrence of Apical Dentinal Microcracks
in Procedures up to Root-end Filling
Shuntaro NAKAYAMA, Taro NISHIDA, Miki SEKIYA, Munehiro MAEDA and Masaru IGARASHI （ 1 ）Differentiation of Murine Enamel Organ-derived Tissue Stem Cells
into Cementoblasts after Transplantation
Minoru TAKASE, Naoki MARUO, Kazuhiko OKAMURA, Nanako TSUCHIMOCHI, Hiroaki YAMATO,
Kimiko OHGI, Atsushi NAGAI, Takashi KANEKO, Yasunori YOSHINAGA and Ryuji SAKAGAMI （ 12 ）Five-year Prognosis and Risk Factor Analysis of Open Flap Debridement for Older People
Yukari AOKI-NONAKA, Takahiro HOKARI, Yumi MATSUKAWA, Keisuke SATO, Miki HARA,
Mai TAKEUCHI, Kei TAKAMISAWA, Kyoko YAMAZAKI, Takahiro TSUZUNO, Hikaru TAMURA,
Takumi HIYOSHI, Fumiya MEGURO, Emi HOSHIKAWA, Aoi MATSUGISHI, Chihiro KANEKO,
Shuhei MINEO, Moe YAMASHITA and Koichi TABETA （ 22 ）Shaping Ability of Controlled Memory Wire Nickel-titanium Files by Minimally Invasive Endodontics
Pil Seoung KANG, Takahito TSUKUDA, Kazuhiro ITONAGA,
Masahito YAMANE, Noriko MUTOH and Nobuyuki TANI-ISHII （ 35 ）Discoloration of Conventional Resin Composite Cements and Self-adhesive
Resin Cements Immersed in Water for 180 Days
Ayaka HORI-ISHIKAWA, Yuka OGAWA-UZAWA, Ayako OKADA, Daichi AIZAWA, Nana SAKAEDA-NAITO,
Hiro MATSUMOTO, Kaoru OHMORI, Masao HANABUSA and Takatsugu YAMAMOTO （ 43 ）Comparison of Shaping Ability between Nickel-titanium Hand Filesand Nickel-titanium Rotary Files during Glide path
Masashi YAMADA, Norio KASAHARA, Satoru MATSUNAGA, Tomoyuki INOSE, Hiroki IWASAWA,
Ryo NAKAJIMA, Yoshiki TAMIYA and Masahiro FURUSAWA （ 52 ）Influence of Additional Insertion after Reaching the Working Length on Root Canal
Transportation and Extrusion of the Gutta-percha Point in Five Ni-Ti Rotary File Systems
with Different Bending Properties
Fumiyasu MURAYAMA, Hirokazu SUGITA, Miki SEKIYA, Keisuke SAIGUSA,
Taro NISHIDA, Munehiro MAEDA and Masaru IGARASHI （ 59 ）Clinical Assessment of Root Caries Treatment Based on the Discolorationof Silver Diamine Fluoride as a Caries Indicator
Yuki MURASE, Hanemi TSURUTA, Tomohiro TAKAGAKI, Shusuke KUSAKABE,
Masaomi IKEDA, Michael F BURROW and Toru NIKAIDO （ 73 ）The Effects of Dietary pH Changes on Tooth Staining of the Enamel Surface
Hiroki SUGITO, Takako EGUCHI, Rika TANAKA, Akiko HARUYAMA and Takashi MURAMATSU （ 80 ）Evaluation of the Hardness of Infected Root Canal Dentin with Fluorescence Excitation
Shuya SHIBANO, Mie MAYERS, Tokuji HASEGAWA, Takashi TAKAKI and Katsuhiko ISATSU （ 86 ）Publishedby
THE JAPANESE SOCIETY OF CONSERVATIVE DENTISTRY（JSCD）
c/o Oral Health Association of Japan（Kōkūhoken kyōkai）
1‒43‒9, Komagome, Toshima-ku, Tokyo 170‒0003
Japan

Effect of an MDP-based Cleaner on Adhesion to Dentin Contaminatedwith a Temporary Luting Cement
Taiho IDONO, Shojiro SHIMIZU, Tomohiro TAKAGAKI, Michael F. BURROW and Toru NIKAIDO （ 95 ）Color Evaluation of Porcelain Laminate Veneer Restorations
Bonded with a Single-shade Resin Composite
Toshinari TOYAMA, Mikihiro KOBAYASHI, Rintaro SUGAI, Yuiko NIIZUMA and Atsufumi MANABE （ 104 ）Comparison of Root Canal Preparation Ability of Three Gold Wire-based NiTi Rotary File Systems
in Simulated Curved Root Canals
Miki SEKIYA, Shuntaro NAKAYAMA, Fumiyasu MURAYAMA, Kentaro FURUTA,
Yoshiyuki INUYAMA, Munehiro MAEDA, Kazuo KITAMURA and Masaru IGARASHI （ 113 ）2-deoxy-D-glucose Inhibits Cell Activity by Inhibiting Intracellular Glucose Metabolism
in Human Gingival Fibroblasts
Yoichiro TAGUCHI, Hirohito KATO, Runbo LI, Tsurayuki TAKAHASHI, Daisuke KIMURA,
Kosho KASHITANI, Niina MASU, Hitoshi AZUMA, Chizuko OGATA and Makoto UMEDA （ 121 ）Development and Clinical Evaluation of Anti-Aspiration Gauze Roll
Assessment of Water Absorbency and Usability of Gauze Roll―An Exploratory Study
Chiharu KAWAMOTO, Ryotaro YAGO, Mai FUKUYAMA, Toru TANAKA,
Hidehiko SANO and Atsushi TOMOKIYO （ 129 ）Three-dimensional Analysis of the Shaping Characteristics and Ability of a Novel Ni-Ti Rotary File
Kazuma MATSUMOTO, Haruna HIROSE, Kota ISSHI, Seishiro FUJIMASA,
Shingo KANEMARU, Tsukasa YANAGI and Etsuko MATSUZAKI （ 137 ）Inhibition of Intracellular Glucose Metabolism by 2-deoxyglucose Suppresses Proliferation
and Differentiation of Mouse Osteoblast-like Cells
Hirohito KATO, Yoichiro TAGUCHI, Runbo LI, Chiaki MANDAI, Masahiro NOGUCHI, Kei SHIOMI,
Hideki AKIMOTO, Sho MIZUTANI, Tomoko KANDA, Chizuko OGATA and Makoto UMEDA （ 144 ）Case Report
Risk of an Immediate-type Allergy to Minocycline Hydrochloride Ointment
Maki HOSOKI, Yoshifumi OKAMOTO, Toyoko TAJIMA, Mayu MIYAGI, Yoshiaki KUBO,
Maiko SOGAWA, Resmi RAJU, Shaista AFROZ and Yoshizo MATSUKA （ 152 ）Editorial Board of Operative Dentistr y, Endodontology and Periodontology
Editor-in-Chief
Hideki SHIBA

Graduate School of Biomedical and Health Sciences, Hiroshima University

Associate Editor

Yoshihiro NISHITANI
Kagoshima University Graduate School of Medical and Dental Sciences

Editorial Board

Atsushi KAMEYAMA
Matsumoto Dental University

Motohiro KOMAKI
Kanagawa Dental University Faculty of Dentistry

Keiso TAKAHASHI
Ohu University School of Dentistry

Osamu TAKEICHI
Nihon University School of Dentistry

Mamoru NODA
School of Dentistry, Iwate Medical University

Yasushi FURUICHI
School of Dentistry, Health Sciences University of Hokkaido

Keiichi HOSAKA
Tokushima University Graduate School of Biomedical Sciences

Takahiko MOROTOMI
School of Dentistry, Aichi Gakuin University

Kunihiko YOSHIBA
Niigata University Graduate School of Medical and Dental Sciences

Atsutoshi YOSHIMURA
Nagasaki University Graduate School of Biomedical Sciences

Masahiro YONEDA
Fukuoka Dental College

Editorial Secretar y
Katsuhiro TAKEDA

Graduate School of Biomedical and Health Sciences, Hiroshima UniversityDec, 2023

1

Original Article

ODEP 3（1）：1～11，2023

Effect of Apical Limit Settings on the Occurrence of Apical Dentinal
Microcracks in Procedures up to Root-end Filling
Shuntaro NAKAYAMA, Taro NISHIDA, Miki SEKIYA, Munehiro MAEDA and Masaru IGARASHI
Department of Endodontics, The Nippon Dental University School of Life Dentistry at Tokyo

Abstract
Purpose: In this study, teeth in which root canal shaping was performed with different apical limit settings
were used to clarify the timing of development of apical dentinal microcracks（microcracks）. The microcracks occurring during root canal shaping were observed using micro-computed tomography（micro-CT）.
Root canal obturation of the experimental teeth was followed by root-end resection and cavity preparation
for the same teeth, and the occurrence of microcracks on the resected root surfaces was evaluated.
Methods: Nineteen human-extracted mandibular canines and premolars with similar root morphology wereused. Root planting models with exposed apex（test piece）were prepared for the teeth. Once a tooth wasremoved, micro-CT scanning was performed before the procedure. The apical limits were set in threegroups（n＝6）: at the anatomical apical foramen（Af）, at 1 mm inner Af（Af－1）, and at 1 mm outer Af（Af＋1）. After root canal shaping with nickel-titanium rotary files under wetting, micro-CT scans were performed, and the presence of a microcrack was evaluated in each image. After root canal obturation, the apex
was resected perpendicularly to the tooth axis using carbide burs at 3 mm from the apex. The resected root
surfaces were stained with methylene blue solution, and stereomicroscopic observation was performed underlight-emitting diode（LED）transillumination to record the occurrence and direction of microcracks. The
length of microcracks from the root canal wall was also measured. Following these observations, 3 mm depth
root-end cavities were prepared along the root canal axis using an ultrasonic tip. Subsequently, staining andtransillumination observations were performed using the preview method, and the occurrence and directionof microcracks on the resected root surface were recorded. The length of microcracks from the wall of theroot-end cavity was also measured.
Results: Micro-CT images demonstrated no new microcracks during root canal shaping. After root-end
resection, the number and length of microcracks increased in the order of the Af－1, Af and Af＋1 groups.
After root-end cavity preparation, the detected incomplete cracks ran in the labiolingual direction and thecomplete crack ran in the mesiodistal direction. Furthermore, the length of microcracks after root-end cavity
preparation was significantly greater in the Af＋1 group than in the Af－1 group（p＜0.05）.
Conclusion: The results suggest that root canal shaping with apical limit settings outside the root causes thinning of the dentin and increases the probability of microcracks during ultrasonic root-end cavity preparation.
Key words: apical limit settings, apical dentinal microcrack, root-end cavity preparation

Corresponding author: Shuntaro NAKAYAMA, Department of Endodontics, The Nippon Dental University School of Life
Dentistry at Tokyo, 1‒9‒20, Fujimi, Chiyoda-ku, Tokyo 102‒8159, Japan
TEL: ＋81‒3‒3261‒5698, FAX: ＋81‒3‒5216‒3718, E-mail: s-nakayama2119004@tky.ndu.ac.jp
Received for Publication: December 9, 2022/Accepted for Publication: January 6, 2023
J-STAGE advance published date: October 12, 2023
DOI: 10.11471/odep.2023‒001

ODEP Vol. 3, No. 1

2

Introduction

Materials and Methods

The aim of root canal treatment is to obtain a proper

Nineteen human-extracted mandibular canines andprognosis through proper root canal shaping, disinfec-

premolars with similar root morphology, which hadtion and obturation1‒4）. Root canal shaping is performed

been preserved underwater after extraction, were used

to ensure mechanical cleaning and the provision ofin the experiment. The teeth had no caries or resto-

proper morphology for root canal obturation5）. In thisrations in the root region and were not endodonticallycase, mechanical root canal shaping with instruments

treated. The use of human-extracted teeth wasshould be limited to apical constriction6,7）. It has been

approved by the Ethical Committee of The Nippon

reported that if the apical limits are incorrectly set

Dental University School of Life Dentistry at Tokyo

beyond the length of the tooth, excessive instrumenta-（NDU-T2021-25）.

tion can occur, leading to the development of microcracks

8,9）. These microcracks are the starting points offractures. They gradually develop into vertical root
fractures owing to the repeated application of occlusal
force during mastication10‒12）. A vertical root fracture is
the most common cause of tooth extraction; hence, it isAll endodontic procedures were completed by a dentist（with 5 years of clinical experience）.
1 ．Experiment Ⅰ: Effect of over-instrumentationon the roots during root canal shaping
1 ）Preparation of a root planting model with the
apex exposed

important to protect against root fracture13）. In cases

After removing the calculus and soft tissues on thewith poor response to nonsurgical root canal treat-root surface with a Gracey curette（Hu-Friedy, Chicago,ments, endodontic surgery should be used to remove

IL, USA）, the root surface was covered with a single

the cause of the periapical lesions. In particular, root-

layer of aluminum foil. An acrylic tube with an innerend resection and filling can reliably remove the lesion

diameter of 16 mm and a height of 12 mm was filled

and closely seal the root canal at the apex 14‒16）. In

with auto-polymerizing resin（Ostron Ⅱ, GC, Tokyo,

recent years, ultrasonic tips have been used for root-

Japan）and implanted so that the tooth axis was as ver-end cavity preparation17）. Compared with the conven-tical as possible. After the auto-polymerizing resin hadtional method of using a round bur attached to a micro-

cured, the tooth with the aluminum foil was removed.

motor handpiece, it is possible to prepare root-end cavi-

To ensure the adherence of a soft material correspond-ties along the long axis of the tooth with less risk ofing to the periodontal ligament to the tooth when theperforation 18,19）. However, microcracks may occur

tooth was reimplanted, an adhesive material（Fusion Ⅱ

20）

during root-end cavity preparation .

adhesive, GC）was applied to the inner surface of thePrevious reports on the occurrence of microcracks at

auto-polymerizing resin. After drying, a self-wetting

the root apex have been evaluated based on the apicalhybrid polysiloxane impression material（Fusion Ⅱ

limit settings of the root canal shaping; however, no

extra wash type, GC）was filled, and the tooth wasstudy has evaluated the effect of root-end resection and

immediately reimplanted25）. After the impression mate-ultrasonic root-end cavity preparation on microc-

rial was cured, the tooth was removed, and the basal 3

racks8,21‒24）. Therefore, in this study, teeth in which root

mm of the tube and resin were polished and removed

canal shaping was performed with different apical limit

under water cooling using silicon carbide grinding

settings were used to clarify the timing of microcrack

paper（#80 and #400, Buehler, Lake Bluff, IL, USA）.

development. The microcracks occurring during root

After the root planting models（test piece）were pre-canal shaping were observed and evaluated nonde-

pared to expose 3 mm of the root apex（Fig. 1）, thestructively and longitudinally using micro-computedroots of the teeth were cut under water cooling perpen-

tomography（micro-CT）. The root canal obturation of

dicular to the tooth axis at 12 mm from the root apex

the experimental tooth was followed by root-end resec-

using a low-speed saw（Isomet LS, Buehler）.

tion and ultrasonic root-end cavity preparation for the2 ）Micro-CT scanning before root canal shaping

same teeth, and the occurrence of microcracks on the

Each test piece was scanned using a micro-CT

resected root surfaces was evaluated.

（ScanXmate-D100SS270, Comscantecno, Kanagawa,Dec, 2023

Apical Dentinal Microcracks Related to Preparations

3

Root dentin

w i t h w a t e r 2 8）. A f t e r w a r d s , a n 1 8％ e t h y l e n e d i -Impression
material
Auto-polymerizing resin
Acrylic tube

aminetetracetic acid solution（Ultradent EDTA 18％,
Ultradent）was applied for 1 min. and the canal was
irrigated again with NaClO. The root canal was dried
with paper points（ProTaper Gold Absorbent Point,
Dentsply Sirona）. Micro-CT scans were obtained afterFig. 1

Cross-sectional image of test pieceroot canal shaping, under the same conditions as control imaging.
5 ）Root canal obturation

Japan）, from the root apex to 6 mm under the condi-

Root canal sealer（AH Plus Jet, Dentsply Sirona）wastions of 10.332μm/pixel at 80 kV and 80μA. The

placed into the root canal using a #35 K file. A ProTa-

images were used as control images before root canalper Gold F4 gutta-percha point（ProTaper Gold Con-shaping.

form Fit Gutta-Percha, Dentsply Sirona）, which3 ）Determination of working length

matched the geometry of ProTaper Gold F4, wasA #10 stainless steel hand K file（MANI, Tochigi,

inserted and the root canal was obturated. Subse-

Japan）was inserted into the canal to confirm patency.

quently, excess gutta-percha points were cut at the root

After confirming patency, a #10 K file was inserted into

canal orifice using a heated root canal plugger（Ishizuka,

the canal until the file tip was visible in the anatomical

Saitama, Japan）, then the gutta-percha points wereapical foramen and the length from the coronal refer-lightly pressed in the apex direction and stored in anence plane to the tip of the file was measured. The

incubator at 37℃ with 100％ humidity. After confirm-

experimental teeth were divided into three groups（n＝ing complete setting at 24 hr, micro-CT scans were6）: the apical limits were set at the anatomical apicalobtained after root canal obturation.

foramen（Af）, at 1 mm inner anatomical apical foramen

To confirm whether microcracks could be observed（Af－1）, and at 1 mm outer anatomical apical foramenon micro-CT scans, root canal shaping was performed（Af＋1）.

on another tooth to F4 under Af－1 conditions. A4 ）Root canal shaping

spreader（Star Dental D11T, DentalEZ, Lancaster, PA,

Root canal shaping was performed using nickel-tita-

USA）was placed in the root canal to intentionally

nium rotary files（ProTaper Gold, Dentsply Sirona, Bal-

induce vertical root fracture as a positive control andlaigues, Switzerland）attached to an endodontic torquemicro-CT scans were obtained29）.

control motor（X-Smart IQ, Dentsply Sirona）. ProTaper

6 ）Evaluation

Gold was used because of its improved resistance toThree-dimensional images were reconstructed fromcyclic fatigue and flexibility compared to conventional

tomographic images obtained pre-root canal shaping,nickel-titanium rotary files26,27）. At first, a glide path

post-root canal shaping and post-root canal obturation

was created using ProGlider（#16/.02, Dentsply Sirona）.

using image processing software（TRI/3D-BON, Ratoc

Subsequently, the preflaring was done using SX

System Engineering, Tokyo, Japan）. The area from the（#19/.04）to the coronal third. Root canal shaping wasroot apex to 6 mm（581 cross-sectional images for eachperformed in the following order until the set working

test piece）was defined as the observation site. In eachlength was achieved: S1（#18/.02）, S2（#20/.04）, F1

image obtained by micro-CT scanning, a line continu-（#20/.07）, F2（#25/.08）, F3（#30/.09）and F4（#40/.06）.

ously reaching the external surface of the root from theDuring root canal shaping, a 2.5 ml syringe（Terumo

canal wall was defined as a complete crack and a line

syringe, Terumo, Tokyo, Japan）with a 27 G needle（Jetextending from the canal wall into the root dentin not

Needle Straight type, B. S. A. Sakurai, Nagoya, Japan）

reaching the external surface of the root was defined as

was used to frequently irrigate the canal with 3％

an incomplete crack23）. In addition, the presence or

sodium hypochlorite solution（NaClO: ChlorCid, Ultra-

absence of microcracks was evaluated.

dent, South Jordan, UT, USA）and patency was maintained with a #10 K file. During root canal shaping, theroot apex of the test piece was continuously moistenedODEP Vol. 3, No. 1

4

2 ．Experiment Ⅱ: Effect of root-end resection on

2 ）Evaluation of microcracks on the resected root
surfaces after root-end cavity preparationroots in over-instrumented teeth

Similar to Experiment Ⅱ, the resected root surfaces

1 ）Root-end resection
A straight plain fissure carbide bur（#57, VDW,

were stained with methylene blue solution and the sur-Munich, Germany）was attached on an electric hand-faces were recorded and observed under a stereomi-

piece（Ti-Max Z95L, NSK, Tochigi, Japan）and the apicalcroscope with LED transillumination. A line continu-

3 mm was resected to an angle of 90 degrees to theously reaching the external surface of the root from thetooth axis under 30,000 rpm water cooling.

root-end cavity wall was defined as a complete crack

2 ）Evaluation of microcracks on the resected root

and a line extending from the root-end cavity wall into
the root dentin not reaching the external surface of thesurfaces after root-end resection
One ％ methylene blue solution（Muto Pure Chemi-

root was defined as an incomplete crack. Both pre-ex-

cals, Tokyo, Japan）was applied with a microbrushisting microcracks on the resected root surfaces after（Tokuyama Dental, Tokyo, Japan）for 5 s to theroot-end resection with a carbide bur and new microc-

resected root surface following root-end resection, theracks generated by ultrasonic root-end cavity prepara-

surface was washed with distilled water, and excess

tion were evaluated. The presence, direction and length

30）of the microcracks from the root-end cavity wall to thewater was removed with water-absorbable paper.

Subsequently, an LED transilluminator（Microlux2,

end of the microcracks were measured.

AdDent, Danbury, CT, USA）with a 1-mm-diameter3 ）Statistical analysis

fiber light guide（Endo-Light Guide, AdDent）was used

IBM SPSS Statistics for Windows, Version 28.0（IBMto observe the resected root surface. The irradiationCorp, Armonk, NY, USA）was used for statistical anal-

was performed in low power mode（1,500 lux）at a posi-ysis, and the significance level was set at 5％. The Kru-

tion 1 mm from the external surface of the root and 90

skal-Wallis multiple comparison test was used to com-31）

degrees to the tooth axis . Furthermore, the resected

pare the length of each microcrack.

root surface was observed and recorded under a stereomicroscope（Leica MZ75, Leica Microsystems, Ben-

Results

sheim, Germany）. Microcracks on the resected root
surface were evaluated using image analysis software（Leica Application Suite, Leica Microsystems）. Similar

1 ．Experiment Ⅰ: Effect of over-instrumentation
on roots during root canal shaping

to experiment Ⅰ, the occurrence of microcracks in eachIn the positive control with root fracture, micro-CTgroup was recorded. The running direction of each

images clearly demonstrated complete and incomplete

microcrack was captured and classified into four direc-

cracks（Fig. 2）. Overall, 31,374 images taken with 18

tions: mesial, distal, labial and lingual. In addition, thetest pieces exhibited no new microcracks. There waslength from the canal wall to the end of the microcrack

no microcrack on images at 3 mm from the root apex.

was measured.

Representative images obtained pre-root canal shaping,

3 ．Experiment Ⅲ: Ef fect of ultrasonic root-end
cavity preparation on roots in over-instr umented teeth
1 ）Ultrasonic root-end cavity preparation
The test piece after root-end resection was fixed in a

post-root canal shaping and post-root canal obturationfor each group are shown in Fig. 2.
2 ．Experiment Ⅱ: Effect of root-end resection on
roots in over-instrumented teeth
1 ）Microcracks after root-end resection

jig（Japan Mecc, Tokyo, Japan）. Subsequently, a dia-

A complete crack was observed in one test piece inmond-coated ultrasonic tip（E32D, NSK）was attachedthe Af＋1 group（Table 1）. The number of test pieces

to an ultrasonic unit（Varios 2 Lux, NSK）and the root-with microcracks and the number of microcracks

end cavity was prepared under water cooling at aincreased in the order of Af－1, Af and Af＋1 groups.

power recommended by the manufacturer’s instructions（mode E, power 6）. The cavity was made 3 mmdeep along the root canal axis until the gutta-percha
point was removed from the cavity wall.

2 ）Direction of running of microcracks after rootend resection
All the incomplete cracks ran in the labiolingual
direction, and the complete cracks ran in the mesiodis-

Dec, 2023
Pre-rootcanalshaping

Apical Dentinal Microcracks Related to Preparations
Post-root
canal
shaping5

ran in the mesiodistal direction（Fig. 4）.

Post-rootcanal
obturation

3 ）Length of microcracks after ultrasonic root-end
cavity preparation
After ultrasonic root-end cavity preparation, theAf－1

length of microcracks increased in the order of Af－1,
Fracture
positive
control

Af and Af＋1 groups（Fig. 5）. The length of microcracks was significantly longer in the Af＋1 group thanin the Af－1 group（p＜0.05）.

Af

Discussion
The effect of root canal shaping on the occurrence ofroot microcracks is often evaluated by the root sectioning method using a low-speed saw and observation of

Af＋1

the resected surfaces using a stereomicroscope30,32,33）. Ina study by Karataş et al., no microcracks occurred in
the group without root canal shaping using the root
sectioning method. In addition, root canal shaping withRepresentative cross-sectional micro-CT
images obtained during root canal treatment
in each group
Positive control was an artificially induced verticalroot fracture. Arrows：complete cracks, arrowhead：
incomplete crack, scale bar＝500μm
Fig. 2a nickel-titanium rotary file caused more microcracks
than in the no root canal shaping group（control
32）
group）. Shantiaee et al. evaluated the effect of root

canal shaping on the occurrence of microcracks using
the root sectioning method. They observed microcracks
in the root canal shaping and no shaping groups. Since
there was no statistically significant difference in thetal direction.

number of microcracks between the root canal shaping3 ）Length of microcracks after root-end resection

and no root canal shaping groups, it was difficult to

After root-end resection, the length of microcracks

determine whether the microcracks identified wereincreased in the order of Af－1, Af and Af＋1 groups,

caused by root canal shaping34）. In the present study,

with no statistical difference（Fig. 3）.

changes in the internal structure of teeth before and

3 ．Experiment Ⅲ: Ef fect of ultrasonic root-end

after the experiment were evaluated using micro-CT

cavity preparation on roots in over-instr u-scans. This enables non-destructive, longitudinal andmented teeth

three-dimensional observation of test pieces from vari-

1 ）Microcracks after ultrasonic root-end cavity
preparationous directions on a computer. Micro-CT scans are usedin endodontics to evaluate root canal anatomy, root

A complete crack was observed in one test piece incanal shaping and the density of root canal obtura-

the Af＋1 group（Table 2）. The number of test pieces

tion35‒37）. De-Deus et al. evaluated root canal shaping

with microcracks increased in the Af group comparedwith a nickel-titanium rotary file using micro-CT scans,with the Af－1 group. In addition, the number and line

and reported no new microcracks in the roots after rootwith microcracks increased in the Af＋1 group com-

canal shaping38）. In addition, it has been reported thatpared with the Af－1 group. Furthermore, the number

microcracks observed after root canal shaping with aof microcracks increased in the Af＋1 group comparednickel-titanium rotary file were also present before rootwith the Af group.

canal shaping39）.

2 ）Direction of running of microcracks after ultrasonic root-end cavity preparation

No new microcracks occurred when root canal shaping was performed using nickel-titanium rotary files

94.7％ of the incomplete cracks ran in the labiolingualwith different apical limit settings; Af－1, Af and Af＋1.

direction. All the complete cracks in the Af＋1 group

Since microcracks were observed in positive controls

ODEP Vol. 3, No. 1

6
Table 1

Number of teeth and lines of microcracks after root-end resection
Af－1

Teeth
Lines

Complete
0
（0％）
0

Af

Incomplete
1（16.7％）
2Complete0（0％）
0Incomplete
3（50.0％）
6

Af＋1
Complete Incomplete
1
4（16.7％） （66.7％）
1
7

Microcrack length（mm）

Complete cracks：Complete, Incomplete cracks：Incomplete

eral cutting efficiency reduced the load applied to the

2.0

root dentin. Hence, microcracks did not occur.

1.5

In this study, root canal obturation was performed toevaluate the influence of the apical limit settings on

1.0

endodontic surgery. Micro-CT, similar to cone-beamcomputed tomography, uses X-rays, so artifacts due to

0.5
0Fig. 3root canal obturation material can be observed, making
Af－1

Af

Af＋1

Length of microcracks obtained after root-end
resection

the detection of microcracks difficult42‒44）. It has also
been suggested that the lateral condensation method
using a spreader and the vertical condensation method
using a thermoplasticized gutta-percha point significantly impact the root dentin and may result in microc-with obvious root fractures, we can conclude thatracks45,46）. Therefore, in this study, a matched-taper sin-

micro-CT imaging is useful for the non-destructive

gle-cone technique with gutta-percha points matched toobservation of microcracks in the roots. Previous stud-

the size and taper of the nickel-titanium rotary file wasies using the root sectioning method have reported an

employed to reduce the impact of root canal obturation

increase in microcracks as the apical limit settings pro-on the roots47,48）.

gressed outside the root canal8,21,22）. On the other hand,

A complete crack was observed on the resected roota study similar to the present study that evaluated thesurface after root-end resection in the Af＋1 group.

occurrence of microcracks in roots due to root canalRose et al. reported that root canal shaping using ashaping with a nickel-titanium rotary file using

nickel-titanium rotary file with a working length of

micro-CT reported no new microcracks regardless of

Af－1 and root-end resection using a #57 bur on a pre-the location of apical limit settings 23,24）. Bier et al.

molar implanted in the mandible from pigs demon-

reported that more root dentin could be removed withstrated no complete crack on the resected root surface

the 6％-tapered nickel-titanium rotary file that was also

in the group that had root canal shaping performed

used for root canal shaping in this study compared withusing a nickel-titanium rotary file. However, root-end

the 2％-tapered hand file specified for use in the ISO

resection was performed on an over-instrumented toothstandard40）. The absence of microcracks during root

using a #80 K file, and a complete crack was observed49）.

canal shaping, despite using a nickel-titanium rotary file

The present study demonstrated a similar trend towith a large taper was due to the cutting efficiency ofstudies involving organisms, suggesting that the root

the nickel-titanium rotary file. Deari et al. compared theplanting model can set up an environment equivalent tolateral cutting efficiencies of austenitic and martensite

the natural periodontal tissue structures.

nickel-titanium rotary files of the same geometry on

The number of microcracks increased in the Af＋1

bovine-extracted teeth and observed that the marten-group compared with the Af－1 group after root-end

site nickel-titanium rotary file demonstrated signifi-

cavity preparation with an ultrasonic tip. Sachdeva etcantly higher lateral cutting efficiency41）. Under theal. prepared a root-end cavity for mandibular premolars

present experimental conditions, the use of a nickel-tita-

up to apical size #40 with a working length of Af－0.5

nium rotary file with a martensite phase and high lat-using a diamond-coated ultrasonic tip at a mediumDec, 2023

Apical Dentinal Microcracks Related to Preparations
Table 2

7

Number of teeth and lines of microcracks after ultrasonic root-end cavity
preparation
Af－1

Teeth
Lines

Complete
0
（0％）
0

Af

Incomplete
5（83.3％）
13

Complete
0
（0％）
0

Af＋1

Incomplete
6（100％）
11

Complete Incomplete
16（16.7％） （100％）1
14Microcrack length（mm）

Complete cracks：Complete, Incomplete cracks：Incomplete

＊

2.0
1.5
1.0
0.5
0

Af－1

Af

Af ＋ 1

Length of microcracks obtained after ultrasonic root-end cavity preparation＊：p＜0.05

Fig. 5
Image of resected root surface obtained
after ultrasonic root-end cavity preparationin the Af＋1 group
Arrow：complete crack, arrowheads：incomplete
cracks, scale bar＝1 mm

Fig. 4power setting used in this study（mode E, power 6）isa medium power setting in mode E（maximum power
setting: 10）suitable for endodontic treatment. The useof low-power settings in ultrasonic units may be recom-

power setting. The results demonstrated that test

mended to reduce the occurrence of microcracks

pieces with preexisting microcracks occurred andduring ultrasonic root-end cavity preparation. It is also

spread more frequently after root-end cavity prepara-possible that the size of the ultrasonic tip affected the50）tion than those without microcracks . Tawil demon-

number of microcracks in the Af－1 group compared

strated that ultrasonic root-end cavity preparation onwith the Af group. Using an ultrasonic tip with a larger

intact roots does not induce new microcracks but may

diameter than the root canal obturation material might

spread existing ones51）. In this study, microcracks were

have resulted in more cutting of the healthy root dentin

observed after ultrasonic root-end cavity preparation inand more force being applied to the dentin. The results

all groups. This may have been due to the power of thesuggest the need for an appropriately sized ultrasonicultrasonic unit. Layton et al. compared the effect of

tip according to the diameter of the root canal obtura-

ultrasonic unit power on ultrasonic root-end cavity

tion material to reduce the occurrence of microcracks.

preparation and reported that the number of microc-

The present study demonstrated that incomplete

racks per tooth was significantly higher for ultrasoniccracks tend to run in the labiolingual direction afterroot-end cavity preparation at the highest power set-root-end resection and cavity preparation. The results

52）

ting than at the lowest . In addition, Palma et al. per-are similar to those of Palma et al., who observed thatformed root canal shaping to #25 with a working length

incomplete cracks occurred in areas with a wide resid-of Af－0.5, followed by root-end resection and root-end

ual dentin width after root-end cavity preparation53）. In

cavity preparation. Microcracks were reported after

addition, complete cracks were observed in the

root-end cavity preparation using the same ultrasonicmesiodistal directions. These may be related to the root53）

tip and power settings as in the present study . The

morphology. The mandibular single-root canal teeth

ODEP Vol. 3, No. 1

8used in this experiment are generally compressed withover-instrumentation was performed during root canala smaller mesiodistal diameter compared with the labi-

shaping. Therefore, we suggest that all dentists select54）

olingual diameter . The complete cracks were proba-

an appropriately sized ultrasonic tip based on the diam-

bly caused by the force applied to the mesial and distal

eter of the root canal obturation material exposed on

dentin of the thinned compressed root due to root canal

the resected root surface and perform root-end cavity

shaping. Teeth with narrower mesiodistal diameters

preparation without applying excessive force in the set-

are significantly more likely to fracture than those withting of the over-instrumentation during endodontic sur-wider diameters, suggesting that great care should begery.

taken when performing root-end resection and root-end
cavity preparation on teeth with narrower mesiodistal

Conclusion

diameters55）.
Few studies that evaluated the effect of endodontic
surgery on the occurrence of microcracks mentioned
the length of microcracks. Min et al. performed root
canal shaping of a molar to apical size #25 with a work-

From the results of this study, the following conclusions can be drawn.
1．No new microcracks occurred during root canalshaping, regardless of the apical limit settings.

ing length of Af－1, followed by root-end resection with

2．The number and length of microcracks on thea diamond disc and root-end cavity preparation using an

resected root surface following root-end resection

inverted cone bur or an ultrasonic tip with varying

increase in the order of the Af－1, Af and Af＋1

power settings of the ultrasonic unit. Confocal micros-groups.

copy of dentin defects on the resected root surface

3．The number of microcracks after ultrasonic root-

demonstrated that ultrasonic root-end cavity prepara-end cavity preparation was the highest in the Af＋1

tion produced dentin defects ranging from 126.0 togroup.

520.0μm in length regardless of the power setting of

4．The incomplete cracks ran in the labiolingual

the ultrasonic unit56）. However, no study has compareddirection, and the complete cracks ran in the mesiodis-

the length of microcracks that occurred at different

tal direction after root-end resection and ultrasonictime points during endodontic surgery by changing theroot-end cavity preparation.

apical limit settings until now. In the present study, the5．The length of microcracks after ultrasonic root-

length of microcracks increased in the order of Af－1,

end cavity preparation was longer in the order of Af－

Af and Af＋1 groups after root-end resection and cav-

1, Af and Af＋1 groups and was significantly larger in

ity preparation. This may have been because thethe Af＋1 group than in the Af－1 group.

amount of root canal dentin removed increases inaccordance with the taper of the file as the file tip

Conflicts of Interest

reaches the outer part of the root canal, resulting in the

There are no conflicts of interest to disclose regarding
this study.

thinning of the dentin57‒59）. The length of microcracks
was significantly longer in the Af＋1 group than in theAf－1 group after root-end cavity preparation. The
Af－1 group is set at the apical constriction, recommended as the apical limit 6,7,60）. Bergenholtz et al.
reported a significantly lower incidence of periapicallesions in endodontically-treated teeth at the proper apical limit61）. The results demonstrated that the over-instrumentation at Af＋1 reduces the success rate ofendodontic treatment and may induce microcracks
during endodontic surgery, indicating the need for caution in setting the working length during root canalshaping. In addition, while examining an endodontically-treated tooth, it is impossible to determine whether

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ODEP Vol. 3, No. 1

12

Original Article

ODEP 3（1）：12～21，2023

Differentiation of Murine Enamel Organ-derived Tissue Stem Cells
into Cementoblasts after Transplantation
Minoru TAKASE1, Naoki MARUO1, Kazuhiko OKAMURA2, Nanako TSUCHIMOCHI1, Hiroaki YAMATO1,
Kimiko OHGI1, Atsushi NAGAI3, Takashi KANEKO4, Yasunori YOSHINAGA1,5 and Ryuji SAKAGAMI1
1

Section of Periodontology, Department of Odontology, Fukuoka Dental College

2

Section of Pathology, Department of Morphological Biology, Fukuoka Dental College
3

Regional Liaison Center, Fukuoka Dental College

4Center for Oral Diseases, Fukuoka Dental College

5

Oral Medicine Research Center, Fukuoka Dental College

Abstract
Purpose: It has been proposed that some enamel organ-derived cells undergo an epithelial-mesenchymaltransition（EMT）during tooth development, differentiate into cementoblasts, and make acellular cementum.
The primary purpose of this study was to elucidate the mechanisms of cell differentiation by transplanting
apical bud-derived stem cells of the epithelial lineage of tooth embryos into the bony defects around themolars and tracking their dynamics. The secondary purpose of this study was to use those tissue stem cells
as a source of cell transplantation for future periodontal tissue regeneration therapy.
Methods: Apical bud cells from GFP-positive mouse incisors were isolated under a microscope, collected,
dispersed, and transplanted into bone defects created around the molars of wild-type mice, and their dynamics were observed for three months. Undecalcified cryosections were made by Kawamoto’s method, andimmunofluorescence staining was made using antibodies against Osteocalcin（OCN）, Cementum protein 1（CEMP1）, and Cytokeratin 10（CK10）.
Results: GFP-positive cells were observed to settle in the periodontal tissue after four weeks of transplantation. Most of the GFP-positive cells showed OCN positivity throughout the observation period. In 12 weeks,some transplanted cells were aligned along the surface of the cementum, and HE images showed the development of cement matrix-like structures around the cells. Among the transplanted cells, cells in the periodontal space were mostly CK10-positive and CEMP1-negative, while cells on the cementum and near alveolar bone were mostly CEMP1-positive and CK10-negative.
Conclusion: Our observations revealed that some transplanted cells underwent EMT and became cementoblast-like on the root surface and osteoblast-like, where they were incorporated into the alveolar bone. Bothcementoblast-like and osteoblast-like cells were CEMP1-positive. On the other hand, CEMP1-negative andCK10-positive cells were located in the middle of the periodontal ligament space, suggesting that they settled
into the tissue as ERM without undergoing EMT. It is interesting to see how the transplanted cells migrate
and settle or differentiate under the influence of surrounding cells. These results suggest that enamelorgan-derived tissue stem cells may differentiate into osteoblasts and cementoblasts/cementocytes and could
be the source of periodontal tissue regeneration.
Key words: cementoblasts, epithelial-mesenchymal transition, Hertwig’s epithelial root sheath
Corresponding author: Ryuji SAKAGAMI, Department of Periodontology, Department of Odontology, Fukuoka Dental College,2‒15‒1, Tamura, Sawara-ku, Fukuoka 814‒0193, Japan
TEL: ＋81‒92‒801‒0411, FAX: ＋81‒92‒871‒9494, E-mail: sakagami@fdcnet.ac.jp
Received for Publication: November 29, 2022/Accepted for Publication: January 11, 2023
DOI: 10.11471/odep.2023‒002

Dec, 2023

Periodontal Regeneration with Enamel Organ-derived Cells

13

ondary purpose of this study was to use those tissue
stem cells as a source of cell transplantation for future

Introduction

periodontal tissue regeneration therapy. In this study,
The lifelong elongation of rodents’ maxillary andapical bud cells from GFP-positive mouse incisors weremandibular incisors has a unique anatomical structure,isolated under a microscope, collected, dispersed, andwith enamel forming on the labial side and the peri-transplanted into bone defects created in the molars ofodontal ligament starting on the lingual side. The mostwild-type mice. Their fate was observed for threeapical side of the incisors, called the labial cervical loop

months. Cellular characteristics were monitored using

initially, has been named the apical bud by Harada etantibodies against Osteocalcin（OCN）, Cementum pro-al.1）. The apical bud contains a cell population composedtein 1（CEMP1）, and Cytokeratin 10（CK10）as markers

of epithelial tissue stem cells corresponding to humanfor calcification, cementum, and epithelial cells, individu-

enamel organs

1‒3）. Using the apical bud as a graftingally.

material may be a valuable tool for studying cell differentiation.

Materials and Methods

We have already reported the successful development of a technique in which incisor apical bud cells

1 ．Experimental animals

from GFP-positive transgenic mice were harvested

This study was approved by the Fukuoka Dental Col-

under a microscope and transplanted into the incisorlege Animal Experiment Committee, considering animalroot tips of wild-type mice4）. The apical bud cells differ-welfare and in compliance with the Fukuoka Dental

entiated into ameloblasts/Hertwig’s epithelial root

College Animal Experiment Guidelines（Fukuoka Dental

sheaths（HERS）/epithelial cell rests of Malassez（ERM）.

College Animal Experiment Approval No. 20007）.

HERS can stimulate and transform mesenchymal cells

Experimental animals were bred in the SPF Laboratoryof the dental follicle into cementoblasts. HERS eventu-

at the Animal Center of Fukuoka Dental College andally remains in the periodontal ligament to form ERM,

kept on a 12-hour light/dark cycle according to the

5,6）. The functions offacility’s guidelines. Fifty-four 10-day-old C57BL/6-TG

the ERM remaining in the periodontal ligament are（CAG-EGFP）mice（Green Mouse, SLC, Shizuoka,

thought to be diverse, including maintenance of theJapan）transfected with the GFP gene were used asperiodontal space, prevention of root resorption anddonors18,19）. Eighteen 6-week-old male wild-type C57BL/

ankylosis, maintenance of homeostasis of the periodon-

6 mice were used as recipients. Donors were housed

tal ligament, and induction of acellular cementum for-with their maternal mice.

some of which undergo apoptosis

7）

2 ．Cell preparation method from donors

mation .

Recently, it has been proposed that HERS undergoes

Ten-day-old C57BL/6-TG（CAG-EGFP）mice werean epithelial-mesenchymal transition（EMT）during

euthanized by inhalation of isoflurane anesthesia over-

tooth development and differentiates into cemento-

dose, the mandible was removed under a stereomicro-

8‒12）. Much circumstantial evidence supports thisscope, and the incisor teeth, including the root apex,

theory in several in vitro experimental systems. How-

were extracted from the mandible. The teeth were

ever, some studies suggest that there are no or few

then treated in 37℃ saline containing 1,500 PU/mL of

blasts13‒17）HERS cells that differentiate into cementoblasts.

Especially in externally transplanted HERS, it isdispase（Dispase, Gibco, Beverly MA, USA）for 20 minutes, and the apical bud portion was isolated from theunclear whether these cells can generate EMT androot apex（Fig. 1）. The isolated apical buds were thenwhether such cell transplantation can be applied forincubated in 1 mL of cell detachment solution

periodontal tissue regeneration with cementum forma-（Accumax, Innovative Cell Technologies, Inc., San

tion.

Diego, CA, USA）for 10 minutes at room temperature

The primary purpose of this study was to elucidate

and collected by centrifugation after cell dispersion.

the mechanisms of cell differentiation by transplanting

3 ．Method of transplantation to the recipientapical bud-derived stem cells of the epithelial lineage ofUnder general anesthesia with isoflurane, 6-week-old

tooth embryos and tracking their dynamics. The sec-

wild-type mice were fitted with a mouth opening

ODEP Vol. 3, No. 1

14
A

A

B
B

μ

Schematic illustration of a method for collecting apical buds
A：Sagittal section of mouse mandible. The enamelof the mandibular incisor is shown in red. Afterremoval of the mandibular incisor from the mandible,
apical buds and the extending tissue transitioning toenamel epithelium（blue）were harvested.
B：Enamel epithelial tissue containing apical buds
collected from the mandibular incisor.
From the tissue fragment shown in the figure, the portion indicated by the dotted circle was separated as anapical bud.
Fig. 1

device, and two holes of 0.5 mm in diameter and 1.5 mm
in depth were drilled into the periodontal ligament of
the maxillary molar using an engine drill without opening periodontal flap（Fig. 2）. Compression hemostasis

Schematic illustration of a method for creatingbone defects in the recipient
A：Sagittal section model of mouse maxilla. Defects
of 0.5 mm in diameter and 1.5 mm in depth were creFig. 2

ated in two locations on the mesial and palatal sides ofthe mouse maxillary first molar using a round bar
attached to a straight handpiece.
B：The palatal surface of the maxillary first molar
undergoing bone defect formation. Drilling was performed at two locations, the mesial surface of themesial root and the furcation area between the mesial
and lingual roots, as indicated by the dotted circles, andcells collected from the donor were carefully injected.

was applied as needed to ensure adequate hemostasis
after drilling. The transplantation of cells was per-removed and fixed in 4％ paraformaldehyde buffer solu-

formed with a micropipette（Reference 2, Eppendorf,tion.

Hamburg, Germany）with a gel loading tip（124-R-204,

4 ．Preparation of pathological specimens

BIO-BIK, Osaka, Japan）. Cell counts were verified on a

The undecalcified maxillary bone was placed in a

blood cell counter. An average number of 3.0×105 cells

10×10×5 mm cryomold container（Sakura FineTek

were inoculated at one socket. The number of trans-

Cryomold, Osaka, Japan）with the occlusal surface fac-

planted cells was three μL per defect, and each holeing upward and frozen in liquid nitrogen with Cryo-Em-

received cells from three donor sites. Dispersed cells

bedding Medium（SCEM, SectionLab, Hiroshima, Japan）.

were injected into the periodontal ligament and bone

The frozen sections were cut with a tungsten knife at

defect without carrier materials. No sutures were made－20℃ in a cryostat（CM3050S, Leica, Wetzlar, Ger-

after injection. Animals were euthanized at 1, 4, and 12many）at a thickness of 4μm by the Kawamoto’s

weeks after transplantation（n＝6 per week, a total of

method（film transfer method）using the height of the

18 animals）, and the right and left maxillae wereroot furcation of the maxillary first molar as a refer-Dec, 2023

Periodontal Regeneration with Enamel Organ-derived Cells

ence. The specimens were observed by HE staining andimmunofluorescence staining. For immunostaining, an

15

Aanti-GFP chicken antibody（Abcam, Cambridge, UK）
was used as the primary antibody, and an anti-chicken
AlexaFluor 488 antibody（Thermo Fisher Scientific Inc.,
Cambridge, UK）as the secondary antibody to enhance
GFP fluorescence. In addition, six samples at one week,
six at four weeks, and three at 12 weeks were treatedwith anti-OCN antibody followed by anti-rabbit
Alexafluor568 antibody. In the three 12-week samples,anti-CEMP1 and anti-CK10 rabbit antibodies（Abcam）
were used as primary antibodies and anti-rabbit
Alexafluor568 antibody（Thermo Fisher Scientific Inc.）

B

C

D

E

as the secondary antibody. All sections were sealed in
VectorShield with DAPI（Vector Laboratories, Newark,
CA, USA）. Tissue sections were observed with a fluorescence microscope（BZ-X710, Keyence, Osaka, Japan）.

Results
Representative examples of tissue images from 1 to
12 weeks after transplanting cells collected from donor
mice into the first molar area of recipient mice areshown.
1 ．One week after transplantation
Images at one week after transplantation are shownin Figure 3. The low-magnification HE image showed
drilled bone defects on the mesial surface of the maxillary first molar’s mesial root and the lingual root’s
mesial furcation side. The transplanted cells showedgreen fluorescence over a wide area on the mesial surface of the maxillary first molar’s mesial root and the
lingual root’s mesial furcation. Most transplanted cells
strongly expressed OCN one week after surgery,although the HE images did not show calcification.
DAPI images showed that some of the transplanted
cells were in an aggregated state.
2 ．Four weeks after transplantation
Four weeks after transplantation, GFP-positive cells
were observed in and around the periodontal ligament（Fig. 4）
. OCN-positive oval-shaped cells were widelydistributed on the cementum and alveolar bone surfaces, and some GFP-positive cells showed strong OCN
expression. These cells showed cementoblast-like andosteoblast-like morphology on HE images.
3 ．Twelve weeks after transplantation
Images at 12 weeks after transplantation are shown

HE-stained and immunofluorescent-stained
images at one week after transplantation
A：HE image of a horizontal section of the maxillary first molar. A magnified image of the red-framedarea is shown in B. The area of the created bone
defect is shown as a dotted white circle. Mes：mesialroot；Dis：distal root；Lin：lingual root
B：Magnified HE image of the mesial surface of thelingual root. The arrowhead and outlined arrowhead
indicate the transplanted cells. C, D, and E show
immunofluorescent-stained images of the same area.
AB：alveolar bone；PL：periodontal ligament
C：GFP-positive cells are mostly found around thealveolar bone（arrowhead）and in the periodontal ligament space（outlined arrowhead）.
D：OCN-positive cells are found around the alveolarbone and the periodontal ligament space, overlapping
most GFP-positive cells.
E：The merged image of C, D, and DNA stainedwith DAPI shows that some transplanted cells are inan aggregated state；most of the GFP-positive cells are
OCN-positive.
Fig. 3

ODEP Vol. 3, No. 1

16

in Figure 5. GFP-positive cells produced cemento-A

blast-like cells in the periodontal ligament and cement
matrix-like structures around the partitions on HE
images. GFP-positive cells were also found along thecementum surface of the root and on the alveolar boneside across the periodontal ligament, which was
OCN-positive. Some GFP-positive cells were also foundin the periodontal space, and even though they were
OCN-positive, they did not show calcification on the HE
images.
4 ．CEMP1-and CK10-positive cells at 12 weeks

BC

after transplantation
Images at 12 weeks after transplantation are shownin Figure 6. Some of the GFP-positive cells migrating
into the periodontal ligament were found. In addition,some of the cells were aligned along the surface of thecementum, indicating CEMP1 positivity, and HE images
showed the development of cement matrix-like structures around the cells. Cells in the periodontal ligamentDE

were mainly CK10-positive and CEMP1-negative, while
many CEMP1-positive and CK10-negative cells were
found on the cementum and alveolar bone surfaces.

Discussion
In our previous study, we transplanted the apical tissue of the mandibular incisor, including epithelial andHE-stained and immunofluorescent-stained
images four weeks after transplantation
A：HE image of a horizontal section of the maxillary first molar. A magnified image of the red-framedarea is shown in B. The area of the created bone
defect is shown as a dotted white circle. Mes：mesialroot；Dis：distal root；Lin：lingual root
B：Magnified HE image of the distal surface of themesial root. The arrowhead and outlined arrowhead
indicate the transplanted cells. C, D, and E show
immunofluorescent-stained images of the same area.
AB：alveolar bone；PL：periodontal ligament；CE：cementum；DE：dentin
C：GFP-positive cells are partly aligned along thecementum surface（arrowhead）and partly in the periodontal ligament space close to the alveolar bone（outlined arrowhead）.
D：OCN-positive cells are found on the cementum
and alveolar bone.
E：Merged image of C, D, and DNA stained withDAPI showing that most of the GFP-positive cells are
OCN-positive.
Fig. 4

mesenchymal cells derived from GFP mice, into wildtype mice and observed the dynamics of the cells by
GFP green fluorescence4）. In the current study, to further limit the number of cells to be transplanted, we
performed enzyme treatment on the grafts and transplanted only cells dispersed from the isolated apicalbuds. With this new approach, we aimed to elucidate
the fate of HERS cells after tissue transplantation andto utilize the cells in new cell-based regenerative therapies.
In periodontal tissue regeneration therapy, thecementum, the periodontal ligament, and the alveolarbone are expected to be regenerated. This is because
the cementum is calcified periodontal tissue bound to
the dentin, and one end of the primary fibers of theperiodontal ligament enters the cementum as Sharpy
fibers, while the other end joins the alveolar bone tohelp hold the tooth in place. This study investigated the
kinetics of epithelial tissue stem cells after transplantation by tracking GFP fluorescence.

Dec, 2023

Periodontal Regeneration with Enamel Organ-derived Cells

17

A

B

C

HE-stained and immunofluorescent-stained images at 12
weeks after transplantation
A：HE image of a horizontal section of the maxillary firstmolar. Magnified images of the red-framed areas are shown in B
and F. The area of the created bone defect is shown as a dottedwhite circle. Mes：mesial root；Dis：distal root；Lin：lingual root
B：Magnified HE image of the distal surface of the mesial root
（palatal side）. The outlined arrowhead indicates the transplanted
cells. C, D, and E show immunofluorescent-stained images of thesame area. AB：alveolar bone；PL：periodontal ligament；CE：cementum；DE：dentin
C：GFP-positive cells are aligned along the cementum surface（outlined arrowhead）.
D：OCN-positive cells are aligned along the cementum surface（outlined arrowhead）.
E：Merged image of C, D, and DNA stained with DAPI showing that most of the GFP-positive cells are OCN-positive.
F：Magnified HE image of the distal surface of the mesial root
（buccal side）. The arrowhead indicates the transplanted cells.
Immunofluorescent-stained images of the same area are shown
in G, H, and I. AB：alveolar bone；PL：periodontal ligament；
CE：cementum；DE：dentin
G：GFP-positive cells are aligned along the cementum surface；some are found in the periodontal ligament space（arrowhead）.
H：OCN-positive cells are aligned along the cementum surface
and in the periodontal ligament space（arrowhead）.
I：Merged image of G, H, and DNA stained with DAPI showing that GFP-positive cells in the periodontal ligament space are
predominantly OCN-positive.
Fig. 5

D

E

F

G

H

I

CEMP1 is explicitly expressed in cementoblasts. It is

The results showed that the cells migrated into thealso weakly expressed in mesenchymal cells such as

periodontal ligament space and differentiated into peri-

20,21）

odontal and cementoblast-like cells. Some of the cells

osteoblasts, osteocytes, cement cells, and fibroblasts.

ODEP Vol. 3, No. 1

18
Fig. 6A

CEMP1- and CK10-positive cells
at 12 weeks after transplantation.

A：HE image of a horizontal section of

B

C

D

E

G

H

I

J

F

K

the maxillary first molar. A magnified
image of the red-framed areas is shown in
B and G. The location of the created bone
defect is shown as a dotted white circle.
Mes：mesial root；Dis：distal root；Lin：
lingual root
B：Magnified HE image of the mesial
aspect of the lingual root（palatal side）.
The arrowhead indicates the transplanted
cells. Immunofluorescent-stained images ofthe same area are shown in C, D, E, and F.
AB：alveolar bone；PL：periodontal ligament；CE：cementum；DE：dentin
C：G F P - p o s i t i v e c e l l s a r e m o s t l yaligned along the cementum surface（arrowhead）and alveolar bone surface.
D：CEMP1-positive cells are found on
the cementum and alveolar bone surfaces.
E：Merged image of C, D, and DNA
stained with DAPI showing that parts of
the GFP-positive cells along the cementum surface are CEMP1-positive.
F：CK10-positive cells are primarilyaligned in the periodontal space.
CEMP1-positive cells（arrowhead in D）
along the cementum surface are found to
be CK10-negative in F.
G：Magnified HE image of the mesial
aspect of the lingual root（labial side）. The
outlined arrowhead indicates the transplanted cells. Immunofluorescent-stained
images of the same area are shown in H, I,
J, and K. AB：alveolar bone；PL：periodontal ligament；CE：cementum；DE：dentin
H：GFP-positive cells are mostly aligned
along the cementum surface（outlined
arrowhead）and alveolar bone surface.
I：CK10-positive cells are found on the
cementum and alveolar bone surfaces.
J：Merged image of H, I, and DNA
stained with DAPI showing that among
GFP-positive cells, those within the periodontal ligament are predominantly
CK10-positive.
K：CEMP1-positive cells are mostlyaligned along the cementum surface.
CK10-positive cells（outlined arrowhead in
I）in the periodontal space are found to be
CEMP1-negative in K.

Dec, 2023

Periodontal Regeneration with Enamel Organ-derived Cells

19

were OCN-positive and CEMP1-positive on the surface

calcified nodules. HERS cells also expressed markers ofof the cementum with cement matrix-like structures.

cementum- and bone-related proteins such as enamel

This experiment observed the healing process inprotein, cytokeratin, vimentin, and E-cadherin. Further-observation periods of 1, 4, and 12 weeks. The trans-

more, TGF-β1-treated HERS cells activated the PI3K/

planted cells were already OCN-positive at one week

AKT pathway, resulting in EMT, and subcutaneous

postoperatively, indicating that a shorter observationtransplantation of HERS cells after 48 hours of TGF-β1

period is needed to observe EMT of enamel organ-de-treatment in mice revealed cementum-like calcification.

rived cells. It was deemed necessary to keep the trans-

Akimoto et al.11） examined gene and protein expres-planted cells over a long period to determine how theysion in vitro using HERS-derived cell lines. The results

are taken up and function in the tissue.

showed that mesenchymal markers such as vimentin

Our observations revealed that some transplanted

and N-cadherin and epithelial stem cell markers such as

cells underwent EMT and became cementoblast-like oncytokeratin14, E-cadherin, and p63 were observed. In

the root surface and osteoblast-like, where they wereaddition, stimulation with TGF-β captured many mes-

incorporated into the alveolar bone. Both cemento-

enchymal markers and EMT markers such as snail1

blast-like and osteoblast-like cells were CEMP1-positive.

and snail2. These results indicate that HERS cells may

On the other hand, CEMP1-negative and CK10-positive

undergo EMT during root formation.

cells were located in the middle of the periodontal liga-

Huang et al.12） investigated cementum formation inment space, suggesting that they settled into the tissuemolars by genetically engineering a mechanism by

as ERM without undergoing EMT. It is interesting towhich keratin14-positive HERS cells maintained βgal

see how the transplanted cells migrate and settle orpositivity after EMT in experiments using K14-Cre;differentiate under the influence of surrounding cells.

R26R mice. They found numerous βgal-positive cells in

These results suggest that enamel organ-derived tissue

the cementum near the root apex and concluded thatstem cells can differentiate into osteoblasts and cemen-

HERS cells had differentiated into cementoblasts.

toblasts/cementocytes and could be the source of peri-

Huang et al.’s study strongly suggests that acellularodontal tissue regeneration.

cementum may be composed of cells derived from the

Numerous in vitro and in vivo experiments support

enamel epithelium.

that HERS undergoes EMT and differentiates into

Thus, many studies have shown that HERS cells may

cementoblasts8‒12）. The first report was by Webb et al.8）

differentiate into cementoblasts. It has also been sug-in 1996, who showed immunohistochemical co-localiza-

gested that different mechanisms by cells of other ori-tion of keratin and vimentin in cementoblasts during

gins may form cellular cementum and acellular cemen-acellular cementum formation in rat molars. Theytum.

assumed that these were HERS-derived and that acellu-

Furthermore, when we look at the early stages oflar cementum could be made by HERS-derived cells.

root development, there is an intermediate cementum

Zeichner-David et al.9） isolated HERS cells from theat the interface between the dentin and cementum sur-molars of genetically modified mice and createdfaces, produced by cells derived from the enamel epi-

HERS-derived cell lines. These cells initially secreted

thelium. This suggests that HERS cells can deposit

enamel-associated proteins such as ameloblastin but

cemental matrix and are likely to differentiate into

gradually changed morphology to secrete an extracellu-

cementoblasts.

lar matrix with calcification that resembled the struc-On the other hand, some studies suggest that HERS

ture of acellular cementum. Based on this finding, theycells do not become cementoblasts. Although the

hypothesized that acellular cementum is formed frominvolvement of HERS in the odontoblast during root

HERS-origin cementoblasts and that cellular cementum

development is widely accepted, it is still controversial

is formed from neural crest-origin cementoblasts.

that HERS cells undergo EMT and differentiate into

immortalized human HERS cells,

cementoblasts. Luan et al.13） used cytokeratin14 as aestablished them as cell lines, and investigated theirmarker of HERS/ERM cells to observe the root sur-properties in vitro. They found that HERS cells could

faces of mouse molars. They found many cytokera-

differentiate periodontal ligament stem cells to produce

tin14-positive epithelial cells in the cementum, which

Sonoyama et al.

10）

ODEP Vol. 3, No. 1

20

they attributed to the encapsulation and incorporation

are observed as fibroblasts in the tissue due to theof HERS cells into the calcifying cemental matrix

EMT of enamel organ-derived epithelial cells.

rather than their differentiation into cementoblasts.

In this study, animal experiments were conducted in

Although most ERM cells remain close to the cemental

mice to elucidate the role of enamel organ-derived cells

surface, they are not strongly involved in cementum

in periodontal tissues and to apply them to periodontal

formation. Their study supports Lester’s view that epi-

tissue regeneration therapy by transplanting them

thelial cells on the root surface may be bound to thedirectly into wounds. The cells may have a dual func-

cementum14）.

tion by becoming cementoblasts and as HERS, inducing
15）

Hirata and Nakamura

reported the absence of ker-

atin-osteopontin co-localization or keratin-bone sialoprotein co-localization in cellular cementum formation

blasts by releasing enamel matrix proteins.
It has been reported that the transcription factor

also

sox2 regulates apical bud cells 24）, and HERS cells

performed immunohistochemical staining on tissue sec-

express genes related to epithelial stem cells andtions of rat molars and found no evidence of keratin-vi-

embryonic stem cell markers such as Oct-4, Nanog, andmentin co-localization or Runx2-keratin co-localization

SSEA-425,26）, so there is a possibility that these cells canamong cementoblasts and no evidence of epithelial cells

be used for tissue regeneration. However, there is cur-differentiating into mesenchymal cells or calcifying16,17）.

rently no suitable cell source for clinical use, and seek-

The keratin-positive cells in the cementum were noting such a source from extracted teeth, ES cells, or iPS

considered cementoblasts/cementocytes, but HERS

cells, is a high hurdle. However, periodontal tissues

using mouse molars. Yamamoto and Takahashi

16）

surrounding mesenchymal cells to become cemento-

cells were left behind in the cementum during cemento-

have distinct cell populations, such as ERMs, which are

genesis12,13）. They concluded that HERS cells do not dif-essential in cementum homeostasis and repair. There is

ferentiate into cementoblasts regardless of whether

potential to develop periodontal tissue regeneration

they are in cellular or acellular cementum and that cells

therapy using these cell sources27）.

in the dental follicle differentiate into cementoblasts, as
has been the conventional theory.

One problem of our study is that the bone defects
were too significant compared to those in experimental

As described above, in vivo studies have yet to reachanimals, resulting in excessive invasion. Thus, thea clear conclusion. This is partly due to the limitations

results were uneven as a controlled study for tissueof the research methodology. Most in vivo studies areregeneration. In the future, we would like to further

based on observations at a single time and do notverify the results by performing cell transplantation

employ a method to follow cells over time to identify

with palatal flap formation only, without creating a bone

12）

HERS cells. We can conclude from Huang et al . thatacellular cementum is produced from HERS cells, atleast in mice.

defect.

Conclusion

In periodontal tissue regeneration therapy using

It was suggested that enamel organ-derived tissue

enamel matrix protein, which is currently used in rou-

stem cells may differentiate into osteoblasts and cemen-tine dental practice, the role of enamel organ-derived

toblasts/cementocytes and could be the source of peri-cells present in the tissue in regeneration is not wellodontal tissue regeneration.

understood; Hammaström et al. reported that there isacellular cementum deposition by the combination of

Acknowledgments

enamel matrix proteins in rat molars and monkey ante-

We would like to express our deep appreciation to Dr.
Hidemitsu Harada, Professor of Iwate Medical University
School of Dentistry, who kindly taught us how to collect apical bud cells from the jaws of mice.

rior teeth22,23）. They considered that mesenchymal cells
differentiated into cementoblasts and formed acellular
cementum by the action of enamel matrix protein.
However, it has not yet been demonstrated that mesenchymal cells are responsible for developing acellular

Conflict of Interest

cementum in periodontal tissue regeneration therapy. It

We have no conflicts of interest to disclose regarding thepresent paper.

is also possible that the mesenchymal cells themselvesDec, 2023

Periodontal Regeneration with Enamel Organ-derived Cells

2114）Lester KS. The incorporation of epithelial cells by

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ODEP Vol. 3, No. 1

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Original Article

ODEP 3（1）：22～34，2023

Five-year Prognosis and Risk Factor Analysis of Open Flap Debridement
for Older People
Yukari AOKI-NONAKA, Takahiro HOKARI, Yumi MATSUKAWA, Keisuke SATO, Miki HARA,
Mai TAKEUCHI, Kei TAKAMISAWA, Kyoko YAMAZAKI, Takahiro TSUZUNO, Hikaru TAMURA,
Takumi HIYOSHI, Fumiya MEGURO, Emi HOSHIKAWA, Aoi MATSUGISHI, Chihiro KANEKO,
Shuhei MINEO, Moe YAMASHITA and Koichi TABETADivision of Periodontology, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University

Abstract
Purpose: Examination of the effectiveness of periodontal surgery for older people for which no solid evidence is available is important because the number of healthy, independent older patients is increasing annually. This study aimed to clarify the risk factors associated with the efficacy and prognosis of open flap
debridement for older patients.
Methods: Clinical data of 186 patients who underwent open flap debridement and were subsequently followed by supportive periodontal therapy（SPT）or maintenance for ＞5 years were retrospectively reviewed.
Patients were divided into two groups according to age at the time of periodontal surgery: an adult group,
aged 20‒64 years and an older group, aged ≥65 years. Improvement, recurrence, and tooth loss were evaluated, and related risk factors were analyzed by multivariate logistic regression analysis.
Results: The improvement of periodontal disease by open flap debridement in the older group was similar
to that in the adult group, and no difference was found in the frequency of tooth loss in the 5 years after thestart of SPT. However, the risk of recurrence at 5 years after the start of SPT was three times higher in theolder group than in the adult group, suggesting that age is a risk factor for recurrence. In this study, no risk
factors were specific to the older group other than age. This suggests that aggressive open flap debridement
can be considered even in patients aged ≥65 years if there are no underlying diseases or compliance problems. However, the possibility of future hospital visits affected by health status and social background, presence of bleeding during probing, increase in pocket depth of ≥6 mm, and changes in the total periodontal
inflamed surface area（total PISA）, should be fully considered, and regular maintenance should be continued
after surgical procedures.
Conclusion: The results of open flap debridement in patients aged ≥65 years were comparable to those inpatients aged ＜65 years. In older patients without underlying disease or compliance problems, open flap
debridement should be actively considered. However, age is a risk factor for recurrence and regular postoperative management should be continued with attention to changes in clinical parameters.
Key words: older people, open flap debridement, risk factor

Corresponding author: Koichi TABETA, Division of Periodontology, Faculty of Dentistry & Graduate School of Medical and
Dental Sciences, Niigata University, 5274, Gakkocho-dori 2-bancho, Chuo-ku, Niigata 951‒8514, Japan
TEL: ＋81‒25‒227‒2871, FAX: ＋81‒25‒227‒0808, E-mail: koichi@dent.niigata-u.ac.jp
Received for Publication: December 14, 2022/Accepted for Publication: February 1, 2023
DOI: 10.11471/odep.2023‒003

Dec, 2023

Five-year Prognosis and Risk Factors of Open Flap Debridement for Older People

23

gical procedures for older patients by retrospectively

Introduction

examining clinical data in healthy patients without periodontitis-related systemic diseases, with the type of

The population aged ≥65 years in Japan was 36.21

periodontal surgery limited to open flap debridement.

million in 2021, with an aging rate of 28.9％, and theaging rate will increase to approximately 40％ byMaterials and Methods

1）

2060 . The number of remaining teeth among older
people has increased markedly, and the proportion of

1 ．Participant selection

those with ≥20 teeth at age 80 years now exceeds

This study was conducted as a longitudinal case-con-50％. However, the prevalence of periodontal diseases istrol study. Data were analyzed retrospectively fromincreasing annually as the number of remaining teeth

medical record information and examination data. The
quality rating（Newcastle-Ottawa Scale9,10））of the obser-

2）

increases .

Periodontal disease is a chronic inflammatory diseasevational study methodology was 7 points.

caused by bacterial biofilm. Periodontal surgical treat-

The study participants were 186 patients（77 men

ment is used when the cause cannot be adequately

and 109 women）. At the first visit, the mean（±SD）age

removed by basic periodontal treatment, and deep peri-

was 54.3±10.9 years, and the mean（±SD）number ofodontal pockets remain.

remaining teeth was 25.1±3.8. These patients visited

Many studies have examined bone defect morphol-

the Division of Periodontics, Niigata University Medicalogy, materials, and surgical techniques as factors

and Dental Hospital between April 1, 1998 and Novem-

involved in the prognosis of periodontal surgical treat-ber 30, 2018, underwent open flap debridement, andment3）. However, no solid evidence has been obtained

then ≥5 years had elapsed after the start of SPT or

as to whether periodontal surgical treatment can be

maintenance. The participant selection process isequally effective in older people. The health status andshown in Figure 1. In addition, selection criteria wereindependent capacity of older people vary widely, andage of ≥20 years at the first visit, presence of peri-

their dental treatment is often limited by their underly-odontitis, and absence of systemic diseases related toing disease. Therefore, the goals of periodontal treat-

periodontitis（diabetes and cardiovascular diseases）. Of

ment for older people are unclear, making it difficult to

the 85 patients excluded because of having ＜5 years ofdesign a study with a large-enough sample size to mini-

SPT or maintenance, 81 had ＜5 years postoperatively
as of November 30, 2018, and 4 were excluded because

mize bias.
Maintenance and supportive periodontal therapy

they discontinued treatment during SPT or maintenance.

（SPT）are important for maintaining dental and oral

The open flap debridement was performed after

function after periodontal surgical treatment, and thebasic periodontal treatment when the probing pocket

lack of compliance is significantly associated with tooth

depth was ≥4 mm. The surgeon suggested open flap

loss4,5）. However, older people often discontinue regulardebridement on the basis of factors, including bone

visits because of various psychological, physical, anddefect morphology. Informed consent from the patientssocial reasons. For example, older patients with demen-

was obtained. Periodontal pocket depths were mea-

tia are less motivated to perform oral hygiene and havesured using the 6-point method and probing was per-6）

more periodontitis , and patients with rheumatoid

formed by a dentist or dental hygienist on our staff.

arthritis and stroke have decreased mobility7,8）. On theOpen flap debridement was performed by a dentist

contrary, healthy life expectancy exceeds 70 years forwith a minimum of 1 year of clinical experience.

both men and women, and many older people leadIn this study, the older group included participants

healthy and independent lives. Thus, the pros and cons

aged ≥65 years, which is the current standard in manyof aggressive dental treatment should be considered,developed countries, including Japan, and the World

including periodontal surgery, considering the health

Health Organization11）. The participants were divided

status and compliance of the individual.

into two groups according to age at the time of peri-

Therefore, this study aimed to clarify the factors thatodontal surgery: the adult group included those aged

influence the efficacy and prognosis of periodontal sur-

20‒64 years, and the older group included those aged

ODEP Vol. 3, No. 1

24
Periodontitis patients

331 persons‒visited the periodontics department from April 1998 to November 2018‒having a history of open flap debridement‒at least 20 years old‒without systemic diseases related to periodontitis
Less than 5 years SPT or maintenance 85 persons
Periodontitis patients‒followed by SPT or maintenance for more than 5 years

246 persons

Data incomplete 60 persons
Final subject periodontitis patients

186 persons

Fig. 1 Process of selecting the participants
Finally, 186 people were targeted in this study.

≥65 years. In this study, 38 participants were aged ≥％）, percentage of sites with PD ≥6 mm（PD ≥6 mm,

65 years at the time of periodontal surgery, 11 were ≥％）, total periodontal epithelial surface area（total

70 years, and the oldest was 79 years of age. In cases

PESA, mm2）
, and total periodontal inflamed surface

with open flap debridement at multiple sites, wearea（total PISA, mm2）.

ensured that all the applicable surgeries were performed within the same age group.
This study was planned in compliance with the Dec-

The diagnostic criteria for tooth extraction during
the treatment period were based on Hopeless’ criteria12）.

laration of Helsinki and was conducted in accordance

3 ．Prognostic evaluation at the tooth levelwith the“Ethical Guidelines for Medical and HealthIn the prognostic evaluation at the tooth level where

Research Involving Human Subjects（MEXT/MHLW,

open flap debridement was performed, data on sex,

December 22, 2014, partially revised on February 28,

smoking, bruxism, and denture use were matched to2017）.”It was approved by the Niigata University Eth-the individual level evaluation for each tooth. Compli-

ical Review Committee（Date of approval: December 28,

ance with treatment was evaluated based on data on

2018; Approval no. 2018‒0297）.

recall intervals and regular visits. Tooth types were

2 ．Participant information and clinical parametersclassified into molar, premolar, and front teeth. Age wasdefined as the age at the time of periodontal surgery,

Participant information was examined with regard to

and PD was evaluated in terms of the deepest pocket

the time from the initial diagnosis to the start of SPT

for the target teeth. Changes of the deepest pocket are（year）, recall interval during SPT（times/year）, num-

shown: first visit, end of SRP, start of SPT, and 5 years

ber of patients with smoking habit（smoking, N）, num-

after the start of SPT.

ber of patients with bruxism（bruxism, N）, and numberof patients using dentures（use of dentures, N）.

With reference to the report by Minabe et al.10）, thefollowing three endpoints were defined in this study:

Clinical evaluation was performed on the results of1 ）Improvement of periodontitis（improvement）: A

four visits: initial visit, after scalling root planing（SRP）,

decrease of PD of ≥2 mm at the start of SPT com-

beginning of SPT, and after 5 years of SPT. The clinicalpared with the data after SRP.

parameters at the individual level were as follows: num-2 ）Recurrence of periodontitis（recurrence）: One or

ber of teeth（N）, lost teeth（N）, plaque control record

more of pocket deepening of ≥3 mm, or periodontal（PCR, ％）, site rate of bleeding on probing（BOP, ％）,abscess observed after 5 years of SPT compared withmean probing pocket depth（PD）
for intraoral（PD mean,the data at the start of SPT.

mm）, percentage of sites with PD 4‒6 mm（PD 4‒6 mm,3 ）Tooth loss（loss）: Extraction or loss of a toothDec, 2023

Five-year Prognosis and Risk Factors of Open Flap Debridement for Older People
Table 1

25

Participant information

Total（N＝186） Age＜65（N＝148） Age≥65（N＝38） p-value
Men（N）φ
Women（N）φSmoking status（N）φBruxism（N）φ77（41.4％）

61

16＞0.999109（58.6％）
14 （7.5％）

87
13

22
1

0.472

11
7
3.4±2.554
3.3±2.0

0.176
0.119
0.781

4.1±1.5

3.9±0.7

0.482

16 （8.6％）
Use of dentures（N）
11 （5.9％）
Time from first visit to start of SPT（year）
3.4±2.4（Range 0‒15）
Recall interval（times/year）
4.0±1.4（Range 0‒13）Adult group aged ＜65 vs. older group aged ≥65. Fisher’s exact test was performed for the items indicated as φ,and the Mann-Whitney U test was performed for other items. Values represent mean±SD, ＊：p＜0.05. SPT, supportive periodontal therapy.

surgery. No significant difference was found in the ratio

within 5 years after the start of SPT.
4 ．Statistical analysis

of male and female participants between the two

Fisher’s exact test and the Mann-Whitney U test

groups. No differences were found in smoking, bruxism,

were used to analyze participant information. For clini-or denture use. The mean（±SD）time from the firstcal data at the individual level, Tukey’s multiple com-visit to the start of SPT was 3.4±2.5 years in the adultparisons test was used to analyze changes over timegroup and 3.3±2.0 years in the older group, with nowithin the same group, and the Mann-Whitney U test

significant difference. The recall intervals during SPT

was used for comparisons between the two groups atwere 4.1±1.5 times/year in the adult group and 3.9±

the same time point.

0.7 times/year in the older group, showing no difference

The chi-square test and multivariate logistic regression analysis were used to analyze the association
between prognostic risk factors and improvement,

between the two groups.
2 ．Changes in the clinical parameters over timeat the individual level

recurrence, and tooth loss. A statistically significant p

Tables 2-a and 2-b show the changes in clinicalvalue of ＜0.05 was used. IBM SPSS Statistics for Win-parameter values over time at the individual level fordows version 26.0（IBM Corp., Armonk, NY, USA）wasthe adult group and older group, respectively. Whenused for statistical analysis. Receiver operator charac-

comparing the changes in clinical parameters from theteristic（ROC）curve analysis was used to predict theinitial visit to those after basic treatment, all values,

13）

risk of recurrence of periodontitis using EZR .
＊except for the number of missing teeth, decreased sig-

A prior analysis of power using G Power 3（http://

nificantly after SRP in both the adult and older groups,

www.gpower.hhu.de/）showed a sample size of 32（chi-in which the number of missing teeth increased signifi-square test, effect size 0.5, α error prob 0.05, power（1-cantly in both groups.

β error prob）0.8, degrees of freedom 1）, which is suffi-

A comparison between values after SRP and at thecient to examine sample sizes at the patient and tooth

start of SPT, i.e., before and after periodontal surgery,levels.

showed no significant decrease in PCR in the adultgroup or in PCR, BOP, PD 4‒6 mm, and total PISA in

Results

the older group, but significant changes were noted inother parameters.

1 ．Participant information

In the comparison at each time point between the

The participant information is shown in Table 1.

adult group and older group, no significant difference

Patients were divided into two age groups: 148 were

was noted between the two groups in all items at the

younger than 65 years（adult group）and 38 were 65

first visit, at the end of basic treatment, or at the start

years or older（older group）at the time of periodontalof SPT, including PD mean, percentage of sites with PDODEP Vol. 3, No. 1

26
Table 2

Changes over time in clinical data at the individual levela）Age＜65（N＝148）
5 years after the start of
SPTFirst visit

End of SRP

Start of SPT

Number of teeth（N）

25.3±3.824.4±4.3（＊＜0.0001）

Lost teeth（N）
PCR（％）

2.7±3.833.0±27.0

23.7±4.5（＊0.0001）
4.3±4.5（＊0.0001）

23.1±4.9（＊＜0.0001）
4.9±4.9（＊＜0.0001）

4.0±6.2（＊＜0.0001）

10.4±14.3（0.218）

19.3±20.8

6.7±7.9（＊＜0.0001）

8.0±13.1（0.461）

BOP（％）PD mean（mm）

3.5±1.1

PD 4‒6 mm（％）
PD≥6 mm（％）

28.1±17.7

Total PESA（mm ）Total PISA（mm2）

7.6±11.5

3.6±4.3（＊＜0.0001）
9.3±11.6（＊＜0.0001）
2.7±0.6（＊＜0.0001）

2.3±0.5（＊＜0.0001）
5.9±8.2（＊＜0.0001）

13.5±13.9（＊＜0.0001）
1.8±3.2（＊＜0.0001）

0.3±0.7（＊＜0.0001）＊＊

4.6±6.2（0.897）
2.3±0.5（0.276）
6.2±9.0（0.945）
0.6±1.8（0.136）

1,849.1±741.8

1,282.7±411.5（ ＜0.0001） 1,023.5±297.1（ ＜0.0001） 1,024.7±325.6（0.999）

501.4±604.8

133.5±176.2（＊＜0.0001）

58.9±95.2（＊＜0.0001）

63.7±87.6（0.935）

First visit

End of SRP

Start of SPT

5 years after the start of
SPT

Number of teeth（N）

24.4±4.1

23.6±4.5（＊0.002）

23.0±4.4（＊0.046）

22.4±4.7（＊0.025）

Lost teeth（N）

3.5±4.1

4.4±4.5（ 0.002）

5.0±4.5（ 0.046）

5.7±4.7（＊0.025）

PCR（％）

31.1±29.99.3±11.7（ 0.001）

8.1±11.5（0.995）

11.8±14.1（0.812）

BOP（％）

22.6±26.6

9.0±12.6（0.083）#

PD mean（mm）
PD 4‒6 mm（％）

3.2±0.96

2.7±0.5（＊0.020）

4.5±6.3（0.997）

26.0±18.6

13.6±13.7（ 0.0002）

PD≥6 mm（％）

4.8±6.32b）Age≥65（N＝38）

Total PESA（mm2）
Total PISA（mm2）

＊

＊

＊

5.2±7.4（＊0.004）

2.2±0.5（＊＜0.0001）＊

6.5±14.0（0.085）

1.4±1.9（＊0.003）

2.4±0.4（0.222）
5.6±7.8（0.998）

0.4±0.7（＊0.012）#
1.0±1.6（0.088）#
131.7±177.6（0.073）

1,587.6±581.0

1,238.0±332.9（＊0.0004）

946.5±281.9（＊＜0.0001） 1,003.7±261.6（0.518）

530.3±663.6

92.50±134.2（ 0.0009）

66.9±113.3（0.796）＊

Clinical data at the individual level of（a）the adult group aged ＜65 and（b）older group aged ≥65. BOP, bleeding on
probing；PCR, plaque control record；PD, probing depth；SPT, supportive periodontal therapy；SRP, scaling root planing；Total PESA, total periodontal epithelial surface area；Total PISA, total periodontal inflamed surface area. Values
represent mean±SD.
Tukey’s multiple comparisons test was performed for the successive changes at the following points within the samegroup：（1）first visit vs. end of SRP,（2）end of SRP vs. start of SPT,
（3）start of SPT vs. 5 years after the start of SPT.
＊：p＜0.05. The Mann-Whitney U test was performed to compare the values between the adult group aged ＜65 and theolder group aged ≥65 at each time point. #：p＜0.05

4‒6 mm and PD ≥6 mm. Differences in the BOP positivity rate（p＝0.042）, percentage of sites with PD ≥6ference in the amount of PD within 5 years of SPT was

mm（p＝0.026）, and total PISA（p＝0.033）were found

found between the adult and older groups.

between the two groups at 5 years after the start of

the adult and older groups. Moreover, no significant dif-

When the postoperative course was classified foreach tooth according to the definitions of each item

SPT.
3 ．Frequency of improvement, recurrence, andtooth loss
Table 3 shows data on the 520 teeth that underwent

described in Method 3 above, the results were as follows:1 ）At the start of SPT, 354 teeth showed periodonti-

open flap debridement. The mean PD was significantly

tis improvement, and the improvement rate was 68.1％

deeper in the adult group, and the number of BOP-posi-（adult group, 67.4％; older group, 70.5％）.

tive teeth was also significantly higher in the adult2 ）At 5 years after SPT, 48 teeth showed periodon-

group than in the older group. The mean amount of PD

titis recurrence, and the recurrence rate was 9.4％

reduction from the end of SRP to the start of SPT was（adult group, 7.0％; older group, 18.0％）.

1.95±1.49 mm, with no significant difference between

3 ）During the 5 years of SPT, 7 teeth were lost, andDec, 2023

Five-year Prognosis and Risk Factors of Open Flap Debridement for Older People
Table 327

Subjective tooth information

Total（N＝520） Age＜65（N＝408） Age≥65（N＝112） p-value
Most deep PD（mm）
First visit
End of SRP
Start of SPT
5 years after the start of SPT
BOP positive（N）φReduction of PD from end of SRP tostart of SPT（mm）
Increase of PD from start of SPT to
5 years later（mm）

0.014＊
0.395
0.065
0.566

6.46±1.71

6.56±1.74

6.09±1.52

5.57±1.27
3.62±1.29
3.86±1.52

5.60±1.29
3.64±1.23
3.89±1.61

5.46±1.19
3.53±1.48
4.13±1.94

385

317

68

0.001＊

1.95±1.49

1.96±1.44

1.93±1.680.644

0.32±1.66

0.24±1.48

0.59±2.17

0.347

Subjective tooth information of the adult group aged ＜65 and the older group aged ≥65. Fisher’s exact
test was performed for the items indicated as φ, and the Mann-Whitney U test was performed for otheritems. Values represent mean±SD. ＊：p＜0.05. BOP, bleeding on probing；PD, probing depth；SRP, scaling root planing；SPT, supportive periodontal therapy.

Table 4

Association between the improvement of periodontitis and prognostic risk factors
at the tooth level
Disease category（N＝520）

Variable
Sex
Age（year）
Smoking status
Tooth type

Compliance
Bruxism
Use of dentures
PD（mm）

Improve（N＝354） Non-improve（N＝166）
Men
Women
≥65

＜65
Yes
No
Molar
Premolar
Front teeth
Regular
Irregular
Yes
No
Yes
No
≥6
＜6

p-value

162（45.8％）
192（54.2％）64（38.6％）
102（61.4％）

0.130

79（22.3％）
275（77.7％）58（16.4％）
296（83.6％）
154（43.5％）81（22.9％）
119（33.6％）
340（96.0％）
14 （4.0％）44（12.4％）
310（87.6％）
19 （5.4％）335（94.6％）33（19.9％）133（80.1％）
27（16.3％）
139（83.7％）
79（47.6％）44（26.5％）43（25.9％）161（97.0％）
5 （3.0％）13 （7.8％）
153（92.2％）
3 （1.8％）163（98.2％）

0.569

228（64.4％）44（26.5％）

126（35.6％）

122（73.5％）

1.000
0.202

0.803
0.133
0.064
＜0.0001＊

The chi-square test was performed. Values represent the number of samples（percentage）. ＊：p＜0.05.

the loss rate was 1.3％（adult group, 1.47％; older group,

improvement and risk factors after open flap debride-

0.89％）. There were 2, 2, 2, and 1 missing teeth in thement. The results of the chi-square test showed angroups aged 45‒49, 50‒54, 60‒64, and 70‒74 years,

association between periodontitis improvement andrespectively.
4 ．Association between periodontitis improvement and risk factors
Table 4 shows the association between periodontitis

teeth with a PD of ≥6 mm（p＜0.0001）. Sex, age, smoking, tooth type, compliance, bruxism, and denture usewere not significantly associated with periodontitis
improvement.

ODEP Vol. 3, No. 1

28

Multivariate logistic regression analysis was performed to identify factors associated with periodontitis（p＝0.007, OR＝0.515, CI＝0.318‒0.834）. Age was not
associated with periodontitis improvement.

improvement; the results are shown in Table 5. A PD

Figure 2-a shows the results of the ROC curve analy-of ≥6 mm was significantly associated with periodonti-

sis on the relationship between periodontitis improve-

tis improvement（p＜0.0001, odds ratio［OR］＝5.428,

ment and age at periodontal surgery. Again, no associa-

confidence interval［CI］＝3.561‒9.272）. In addition,

tion was found between age and periodontitis improve-molars were less likely to improve than the front teeth

ment（AUC＝0.535, CI＝0.482‒0.587, sensitivity 0.946,
specificity 0.116）.

Table 5

95％ CI

p-value（Prob＞ChiSq）

Odds
ratio
＜65

1.203
1

0.736‒1.968

0.461

Molar

0.515

0.318‒0.834

0.007＊

Premolar
Front teeth

0.630
1

0.366‒1.084

0.095

Bruxism

Yes
No

1.404
1

0.698‒2.827

0.342

PD
（mm）

≥6

5.428

3.561‒9.272＜0.0001＊

FactorAge（year）
Tooth
type

5 ．Association of risk factors with periodontitis

Multiple logistic analysis of periodontitis improvement and risk factors

≥65

＜6

Table 6 shows the association between periodontitis
recurrence and risk factors at 5 years after SPT. The
results of the chi-square test showed that age（p＝0.001）, tooth type（p＝0.013）, and bruxism（p＝0.028）
were significantly associated with periodontitis recurrence. Sex, smoking, compliance, denture use, and PD
showed no significant association. Furthermore, when
the same sample was divided into adult and older
groups and subjected to the same chi-square test, toothtype and bruxism were not significantly associated withrecurrence in the group aged ≥65 years（Supplemen-1tary Table 1）.
The results of the multivariate logistic regression

Multiple logistic regression analysis was performed.
＊：p＜0.05.

analysis with periodontitis recurrence as the objective

1.0

1.0

0.8

0.8
Sensitivity

Sensitivity

recurrence

0.6
0.4
0.2

1.0

0.8

0.6 0.4
Specificitya62.000（0.695, 0.542）

0.4
0.2

68.000（0.946, 0.116）

0.0

0.6

0.0
0.2

0.0

1.0

0.8

0.6 0.4
Specificity
b

0.2

0.0

ROC analysis：A predictive risk analysis of improvement or recurrence of
periodontitis
The vertical and horizontal axes indicate sensitivity and specificity, respectively.
Age higher than the following cutoff values is considered positive for improvement orrecurrence, and false positives are taken as（1－specificity）.
（a）Improvement of periodontitis and age at surgery（AUC＝0.535, CI＝0.482‒0.587,
sensitivity 0.946, specificity 0.116）. Although the cutoff value for age was set at 68.0
years, this graph shows no correlation between age and improvement.（b）Recurrence of periodontitis and age at surgery（AUC＝0.603, CI＝0.515‒0.691, sensitivity
0.695, specificity 0.542）. The AUC was 0.6, which does not have high predictive
power, but the cutoff value for age at increased risk of recurrence was 62.0 years.
Fig. 2

Dec, 2023

Five-year Prognosis and Risk Factors of Open Flap Debridement for Older People

29

Association between the recurrence of periodontitis and prognostic risk factors at

Table 6the tooth level
Disease category（N＝513）

Variable
Sex
Age（year）
Smoking status
Tooth type

Compliance
Bruxism
Use of dentures
PD（mm）

Recurrence（N＝48） Non-recurrence（N＝465）
Men
Women
≥65

p-value

16（33.3％）
32（66.7％）

205（44.1％）
260（55.9％）20（41.7％）

91（19.6％）

28（58.3％）
11（22.9％）37（77.1％）
31（64.6％）374（80.4％）73（15.7％）
392（84.3％）
197（42.4％）

Premolar
Front teeth
Regular
IrregularYes8（16.7％）9（18.8％）44（91.7％）
4 （8.3％）10（20.8％）116（24.9％）152（32.7％）451（97.0％）
14 （3.0％）46 （9.9％）

0.077

No
Yes
No
≥6

38（79.2％）2 （4.2％）46（95.8％）419（90.1％）
20 （4.3％）445（95.7％）

1.000

29（60.4％）
19（39.6％）

238（51.2％）
227（48.8％）

＜65
Yes
No
Molar

＜6

0.170
0.001＊
0.218
0.013＊

0.028＊

0.230

The chi-square test was performed. Values represent the number of samples（percentage）.
＊：p＜0.05.

Supplementar y Table 1

Association between the recurrence of periodontitis and prognostic risk factors at the tooth levelby groups
Disease category（N＝111）Age≥65

Variable

Sex
Smoking status
Tooth type

Compliance
Bruxism
Use of dentures
PD（mm）

Disease category（N＝402）
Age＜65Recurrence（N＝20）

Non-recurrence（N＝91）

Men
Women
Yes
No
Molar

7（35.0％）13（65.0％）4（20.0％）16（80.0％）12（60.0％）34（37.4％）
57（62.6％）
23（25.3％）
68（74.7％）
36（39.6％）

Premolar
Front teeth
Regular
IrregularYes

4（20.0％）4（20.0％）17（85.0％）3（15.0％）2（10.0％）24（26.4％）
31（34.1％）
86（94.5％）5 （5.5％）14（15.4％）

0.154

No
Yes
No
≥6

18（90.0％）2（10.0％）18（90.0％）77（84.6％）
5 （5.5％）86（94.5％）

0.607

12（60.0％）8（40.0％）44（48.4％）
47（51.6％）＜6p-value
1.000
0.777
0.237

0.732

0.460

Recurrence（N＝28）

Non-recurrence（N＝374）9（32.1％）19（67.9％）7（25.0％）21（75.0％）19（67.9％）

171（45.7％）203（54.3％）50（13.4％）324（86.6％）161（43.0％）4（14.3％）5（17.9％）27（96.4％）
1 （3.6％）8（28.6％）92（24.6％）
121（32.4％）
365（97.6％）
9 （2.4％）32 （8.6％）

20（71.4％）
0 （0.0％）28（100.0％）342（91.4％）
15 （4.0％）359（96.0％）17（60.7％）
11（39.3％）

194（51.9％）
180（48.1％）

The chi-square test was performed. Values represent the number of samples（percentage）. ＊：p＜0.05.

p-value
0.175
0.095
0.039＊

0.518
0.003＊
0.613
0.435

ODEP Vol. 3, No. 1

30

variable are shown in Table 7. Age ≥65 years at peri-

Multiple logistic analysis of periodontitis recurrence

Table 7and risk factors

odontal surgery（p＝0.001, OR＝3.009, CI＝1.598‒5.665）
and bruxism（p＝0.046, OR＝2.221, CI＝1.013‒4.873）
were strong risk factors for periodontitis recurrence.
The molars were more prone to relapse and at higher
risk than the front teeth（p＝0.015, OR＝2.663, CI＝1.212‒5.851）.
Figure 2-b shows the results of the ROC curve analysis on the involvement of age in periodontitis recurrence. The cutoff value for the age at which the risk ofrecurrence increased was 62.0 years（AUC＝0.603, CI＝0.515‒0.691, sensitivity 0.695, specificity 0.542）.
6 ．Association between tooth loss and risk factors
Table 8 shows the association between risk factors
and tooth loss events within 5 years of SPT. The

Odds

Factorratio

p-value（Prob＞ChiSq）

＜65

3.009
1

1.598‒5.665

0.001＊

Molar

2.663

1.212‒5.851

0.015＊

Premolar
Front teeth

1.123
1

0.414‒3.049

0.819

Yes

2.221

1.013‒4.873

0.046＊

No

1

≥6

1.296
1

0.691‒2.430

0.419

≥65

Age
（year）
Tooth
type

95％ CI

Bruxism
PD
（mm）

＜6

Multiple logistic regression analysis was performed.
＊：p＜0.05.

results of the chi-square test showed that age, sex,
smoking, tooth type, compliance, bruxism, denture use,

tal surgery and one that limits periodontal surgery,and PD were not significantly associated with tooth

which later would be relatively chosen more in older

loss.

people with no significant change.
A comparison of data from the start of SPT to 5

Discussion

years at the individual level showed significant changes
in the remaining and missing teeth in both groups, but

1 ．Clinical response to treatment based onno significant differences in other parameters. PCR andchanges in parameters at the individual level

BOP were well below the thresholds for a good progno-

over timesis, indicating that the oral condition of both the adult

Table 2 shows that all clinical parameters significantly changed after SRP in both the adult and older

and older groups was well maintained within 5 years of
SPT.

groups, and no difference was found between the two

In addition, a comparison at each time point between

groups after SRP, indicating that the treatment

the adult and older groups showed no difference for all

response to SRP was similar in the adult and older

items at the initial visit, at the end of basic treatment,groups.

or at the start of SPT. However, the BOP positivity

When the treatment response to open flap debride-

rate, percentage of sites with a PD of ≥6 mm, and total

ment was evaluated by comparing the values after SRPPISA were significantly increased in the older groupwith those at the start of SPT, BOP, PD 4‒6 mm, andcompared with the adult group at 5 years of SPT, sug-

total PISA, which were significantly reduced in thegesting that the risk of periodontitis recurrence or pro-adult group, did not change significantly in the older

gression within 5 years of SPT was increased in thegroup, suggesting that the treatment response to open

older group.

flap debridement in the older group may not be as
marked as in the adult group. However, since these

2 ．Periodontitis improvement at the tooth levelby open flap debridement

examination values were evaluated on the whole oral

Although existing reports have evaluated clinicalcavity, the progress was also influenced by other sites

attachment level（CAL）gain, PD decrease, and bonewhere the open flap debridement was not performed.

level increase in the evaluation of treatment outcomes

Thus, if multiple sites of severe periodontitis were pres-after periodontal surgery3）, since CAL and bone levelent, the frequency and degree of residual inflammation

data were insufficient in this study, we defined

would differ between a treatment strategy that thor-

improvement of periodontitis as a decrease in PD of ≥2

oughly removes inflammation in all sites with periodon-

mm at the start of SPT, i.e., after periodontal surgery,Dec, 2023

Five-year Prognosis and Risk Factors of Open Flap Debridement for Older People

31

Table 8 Association between tooth loss and prognostic risk factors at the tooth level
Disease category（N＝520）

Variable
Sex
Age（year）
Smoking status
Tooth type

Compliance
Bruxism
Use of dentures
PD（mm）

Tooth loss（N＝7） Remaining（N＝513）
Men
Women
≥65

＜65
Yes
No
Molar
Premolar
Front teeth
Regular
Irregular
Yes
No
Yes
No
≥6
＜6

p-value5（71.4％）2（28.6％）221（43.1％）292（56.9％）

0.248

1（14.3％）6（85.7％）1（14.3％）6（85.7％）5（71.4％）1（14.3％）1（14.3％）6（85.7％）1（14.3％）1（14.3％）6（85.7％）0（0.0％）
7（100.0％）111（21.6％）402（78.4％）84（16.4％）429（83.6％）
228（44.4％）
124（24.2％）
161（31.4％）495（96.5％）18（3.5％）56（10.9％）457（89.1％）22（4.3％）491（95.7％）

1.000

5（71.4％）2（28.6％）267（52.0％）246（48.0％）

0.453

1.000
0.359

0.231
0.559
1.000

The chi-square test was performed. Values represent the number of samples（percentage）. ＊：p＜0.05.

compared with that after SRP.

PD reduction in molars was 1.26 mm in areas with PD

In this study, the efficacy of open flap debridement

4‒6 mm, whereas 2.28 mm was observed in areas withshowed improvement rates of 67.4％ in the adult groupPD ≥6 mm16）. In addition, surgical therapy for PD ≥6

and 70.5％ in the older group. The decrease in PD fromthe end of SRP to the start of SPT was 1.96±1.44 mm

mm was more effective than nonsurgical therapy,
resulting in PD reduction of ≥0.6 mm and CAL gain of

in the adult group and 1.93±1.68 mm in the older≥0.2 mm 1 year after treatment, but PD 4‒6 mmgroup, with no significant difference between the two

attachment gain was greater by nonsurgical therapy ingroups（Table 3）. Comparing these values with previ-a systematic review by Heitz-Mayfield et al.17）. In thisous reports, Silvestri et al. reported a decrease of 1.4±

study, periodontitis improvement was defined as PD

1.3 mm in PD 1 year after open flap debridement14）, andreduction of ≥2 mm; thus, the amount of reduction was

Okuda et al. reported a decrease of 2.22±0.81 mm inpossibly small, and improvement was not considered

PD 1 year after surgery as a placebo group for enamelsignificant when the PD was ＜6 mm. In addition, the15）matrix derivatives , which was similar to the reduc-amount of adhesion gained could not be examined intion in the present study. The results of the multivari-this study, but it is necessary to increase the sample

ate logistic analysis also showed no association between

size and examine CAL and bone levels in the future.

age and periodontitis improvement（Table 5）, indicatingthat the adult and older groups had similar treatment

3 ．Risk factors involved in periodontitis recurrence

efficacy with open flap debridement. In this study, theThe analysis of risk factors involved in periodontitis

PCR and compliance of the older group were similar torecurrence showed that age ≥65 years at the time ofthose of the adult group, which may have influenced

periodontal surgery, bruxism, and molars were risk fac-

the treatment effect.

tors（Tables 6 and 7）. The periodontitis recurrence

The results of this study also showed that remaining

rates within 5 years of SPT were 7.0％ in the adultPD ≥6 mm after SRP was significantly associated withgroup and 18.0％ in the older group. The multivariate

periodontitis improvement. Pihlstrom et al. reported

logistic regression analysis showed that the OR ofthat 1 year after open flap debridement, the average

recurrence in those aged ≥65 years was 3.009（TableODEP Vol. 3, No. 1

32

7）, indicating a high risk of periodontitis recurrence inthe prognosis of teeth23）. In this study, the number ofthe older group. The AUC for ROC analysis was 0.6,

missing teeth tended to be slightly higher in the older

which does not have high predictive power, but the cut-group. The effects of loss patterns and presence oroff value for age at increased risk of recurrence was 62

absence of crossbite should be examined in the future.

years（Figure 2-b）
. Similar multivariate logistic regres-In addition, in Table 2, the BOP positivity rate, sites

sion analysis separated by age ≥60 years（p＝0.068,

with a PD of ≥6 mm, and increase in the total PISA

OR＝1.761, CI＝0.958‒3.236）or ≥55 years（p＝0.204,

are clinical indicators of the possibility of recurrence;

OR＝1.495, CI＝0.804‒2.782）showed no significant dif-

therefore, it is necessary to pay more attention toference in either case, suggesting that age ≥65 years ischanges in these values and conduct regular follow-up

associated with recurrence（data not shown）. There-in patients aged ≥65 years and to consider more inten-

fore, the age limit for the elderly was set at 65 years insive plaque control and specialized care for sites at highthis study.

risk of recurrence.

Bruxism showed an OR of 2.2 times higher and4 ．Risk of tooth loss in 5 years of SPT

molars showed a 2.7 times higher risk of recurrence

In previous long-term maintenance studies, the fre-（Table 7）. The prevalence of bruxism is reported to bequency of tooth loss due to periodontal disease has

about twice as high in patients with periodontitis18,19）.

ranged from 1.524） to 9.8％25）, but a review by Cham-

There are also reports that tooth loss is more common

brone et al. reported a loss rate of 6.8％ in a total of 10

19）

in patients with a high likelihood of bruxism . How-

reports9）. In this study, the average loss rate over 5

ever, a recent systematic review concluded that brux-

years of SPT was 1.3％, and the average number of20）

ism does not adversely affect periodontal tissues . The

teeth lost per patient was 0.6, indicating a relatively

simultaneous presence of plaque-induced periodontitis

good prognostic outcome. No risk factors were associ-and bruxism was found to promote connective tissue

ated with tooth loss in this study（Table 8）. Previous

21）

destruction , and thus the control of bruxism is neces-clinical studies on age and periodontal treatment havesary for preventing recurrence after periodontal surgi-

reported a significantly higher risk of tooth loss during

cal procedures.

the maintenance period after periodontal treatment in

As has been reported for tooth types16,22）, open flap

patients aged ＞60 years24,26,27）. However, these studies

debridement showed a greater pocket reduction effectdid not distinguish between periodontal surgical treat-

in nonmolar teeth than in molar teeth. However, sincement or not. On the contrary, reports have not shown afurcation involvement was not evaluated in this study,

relationship between age and tooth loss28,29）. The differ-

further investigation is needed.

ences in the age of participants, severity of periodonti-

Interestingly, neither bruxism nor tooth type wastis, and compliance with treatment in these previous

significantly associated with recurrence when the same

reports were large; further studies are needed to deter-chi-square test was performed only in the group aged

mine whether age is independently related to tooth loss

≥65 years（Supplementary Table 1）, indicating that thefrom other factors9）. In addition, since the rate of tooth

age factor of ≥65 years may be independently associ-loss due to periodontal disease is as high as 7.4％ in

ated with the risk of periodontitis recurrence.

long-term SPT cases of ＞10 years30）, further long-term

The importance of compliance in maintaining peri-

follow-up is necessary.

odontal status during maintenance and SPT has been

5 ．Periodontal surger y in older people

reported4）; there were no significant differences in PCR

The WHO and Japan define older age as age ≥65

or frequency of visits between the adult and older

years. However, the Japan Federation of Gerontologicalgroups. Although the results of this study did notSocieties and the Japan Geriatrics Society have pro-reveal any factors specific to the older group that

posed that the definition of older age be revised to ≥75

increase the risk of recurrence, factors such as theyears based on the incidence of motor function, cogni-

age-related decline in immunity and dietary changes

tive function, disease, and mortality31）, indicating that

may have an effect. According to Lang and Tonetti’s

most older people, especially those aged 65‒74 years,

periodontal disease risk assessment model, the bone-re-maintain their physical and mental health inde-

sorption age ratio and number of lost teeth also affectpendently.

Dec, 2023

Five-year Prognosis and Risk Factors of Open Flap Debridement for Older People

The average life expectancy in Japan in 2021 was

33

aged ≥65 years were comparable to those in patients

81.47 years for men and 87.57 years for women , andaged ＜65 years. In older patients without underlying

the healthy life expectancy is expected to be 72.68

diseases or compliance problems, open flap debride-years for men and 75.38 years for women by 2028, leav-ment should be actively considered. However, age is a

33）

ing a gap of over 10 years . Preventing tooth loss andrisk factor for recurrence and regular postoperative

maintaining masticatory function are extremely import-

management should be continued with attention toant for maintaining quality of life, and periodontal sur-changes in BOP and PD.

32）gery is one option for tooth preservation. In this study,
the number of patients aged ≥70 years was small, with

Conflicts of Interest

11 people in the past 10 years. However, the number of

The authors declare no conflict of interest related to thispaper.

potential periodontal surgical indications is likely to beeven higher considering that the percentage with ≥20
teeth remaining at age 80 is over 50％2）.
The results of this study showed that open flap
debridement had a certain therapeutic effect regardlessof age, with periodontitis improvement being similar inpatients aged ＜65 years and ≥65 years, and that there
was no difference in the risk of tooth loss after 5 years
of SPT. The study participants were an ideal population
who had no health problems and had generally goodcompliance, and the number of remaining teeth and
PCRs at the initial visit, as well as smoking and denture

Acknowledgment
For their great cooperation in conducting this researchand preparing this paper, we thank Dr. Naoki Takahashi,
Dr. Tetsuo Kobayashi, Dr. Noriko Sugita, Dr. Yasutaka
Komatsu, Dr. Tomoki Maekawa, Dr. Haruna Miyazawa, Dr.
Takehiko Kubota, Dr. Kazuhiro Okuda, Dr. Kazuhisa
Yamazaki, and Dr. Hiromasa Yoshie. We also sincerely
thank all doctors of the Division of Periodontology at Niigata
University. This work was supported by the Japan Society
for the Promotion of Science（JSPS; Tokyo, Japan）KAKENHI, grant no. JP19K19021 to T. H.

use, were not different between those aged ＜65 years
and ≥65 years. Furthermore, only four patients
dropped out during the SPT period, and most patientswere under continuous management. Under thesefavorable conditions, open flap debridement may be
actively considered even for patients aged ≥65 years.
Although this study did not examine people aged ≥80years, in patients aged ≥80 years who are sick, thedecision should be made more carefully, not only
because of the risk of inadequate self-care, but also
because of the increased risk of interruption of hospital
visits for health reasons or other reasons.
Further studies are needed to evaluate the efficacy ofregenerative therapy for older people. However, evaluating the attachment level and bone level is necessary,
which could not be included in this study. Collecting
data on cases with long-term follow-up is recommended. Further accumulation of evidence to provide
safer and more effective dental treatment for older people may contribute to extending the healthy life expectancy of the nation.

Conclusion
The results of open flap debridement in patients

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Dec, 2023

35

Original Article

ODEP 3（1）：35～42，2023

Shaping Ability of Controlled Memory Wire Nickel-titanium Filesby Minimally Invasive Endodontics
Pil Seoung KANG, Takahito TSUKUDA, Kazuhiro ITONAGA,
Masahito YAMANE, Noriko MUTOH and Nobuyuki TANI-ISHII
Department of Endodontics, Kanagawa Dental University

Abstract
Purpose: Minimally invasive endodontics（MI Endo）has been advocated on the basis that minimal cuttingof tooth substance prevents root fracture after endodontic therapy. The purpose of this study was to analyze
the shaping ability of thermo-mechanically treated controlled memory（CM）wire nickel-titanium（NiTi）files
using MI Endo.
Methods: Using a J-shaped root canal model, ProTaper Gold（PTG）and ProTaper Ultimate（PTU）files
made of CM wire were used in the experimental group, and ProTaper Universal（PT）files made of conventional NiTi wire were used in the control group, and their cutting properties were compared. The PTU,
PTG, and PT groups were further classified into two subgroups（with and without straight line（SL）access）,
and the root canal width displacement and median displacement were compared between the six groups intotal.
Results: For the PTU and PTG groups, the root canal width displacement on the lateral side slightly
increased at the 1 mm apical measurement, and there was a significant difference between the displacementon the inner and outer sides at 3‒8 mm. However, in the PT control group, the displacement on the innerside increased at 3‒5 mm, and the displacement on the outer side increased at 1 and 8 mm. We compared theroot canal width displacement in the PTU, PTG, and PT groups between those with and without SL access,but no significant differences were observed. Comparison between the SL and no-SL PTU, PTG, and PT
groups revealed no significant differences in root canal width displacement or median displacement at any ofthe measurement sites.
Conclusions: PTU and PTG made with CM wire resulted in accurate root canal formation with no-SL
access.
Key words: minimally invasive endodontics, controlled memory wire, straight line accessCorresponding author: Nobuyuki TANI-ISHII, Department of Endodontics, Kanagawa Dental University, 82, Inaoka-cho, Yokosuka, Kanagawa 238‒8580, Japan
TEL: ＋81‒46‒822‒9527，FAX: ＋81‒46‒822‒9066，E-mail: n.ishii@kdu.ac.jp
Received for Publication: February 6, 2023/Accepted for Publication: March 20, 2023
DOI: 10.11471/odep.2023‒004

ODEP Vol. 3, No. 1

36

ration of anatomically complex curved root canals
because of its lower elastic modulus and higher flexibil-

Introduction

ity and fatigue resistance compared with the austenite
Successful endodontic therapy is based on root canalphase11‒13）. NiTi files for root canal preparation arepreparation that maintains the anatomical root canaltreated with a final surface finishing process in addition

morphology, and the clinical application of nickel-tita-

to heat treatment and mechanical treatment to improvenium（NiTi）files contributes to accurate root canalmechanical properties14）. In 2007, the M wire NiTi alloy

preparation. Root canal preparation is the basic proce-

was developed, and the ProTaper NEXT（Dentsply

dure for removal of cervical dentin, in which the cervi-

Sirona）, in which the austenite phase containing small

cal dentin of the upper third of the root canal is formed

amounts of martensite and the R-layer coexist at body

. Straight line（SL）access facili-

temperature, was applied clinically15‒17）. Controlledtates accurate root canal formation by removing dentin

memory（CM）wire, which was also developed in 2007,

from the root canal orifice and reducing the curvature

has a crystal structure mainly composed of the marten-of the root canal. However, SL access is associated withsite phase as a result of thermo-mechanical treatment,

excessive cutting of cervical dentin, which has been

and has high flexibility at the temperature setting for

into a flared shape

1‒3）

4,5）

reported to reduce tooth fracture resistance. There-clinical use, but as a NiTi file, it does not exhibit super-

fore, in recent years, minimally invasive endodontics

elastic properties18‒20）. The CM wire NiTi files ProTa-（MI Endo）has been advocated, in which root canalper Ultimate（PTU: Dentsply Sirona）
and ProTaper Goldpreparation with minimal cutting of the dentin to pre-（PTG: Dentsply Sirona）, which are the subject of thisserve the orifice dentin is reported to prevent root frac-research, have a martensite transformation start tem-ture7,8）. It has been reported that cervical dentin pres-perature（Ms）and austenite transformation end tem-ervation is more important than coronal occlusal dentin

perature（Af）of 55℃. Both files have been shown to be

6,7）

preservation for the prevention of root fracture.

extremely flexible, as the martensitic state is main-

MI Endo8）is performed mainly on the west coast of

tained at a body temperature of 37℃21‒23）. In addition,

the United States with the objective of minimizing thethe PTU and PTG files are the successors to the Pro-

risk of root canal fracture in comparison with standard

Taper Universal（PT: Dentsply-Sirona）24,25）and perform

root canal preparation. MI Endo advocates maximal

root canal preparation with the same file configuration;

preservation of healthy dentin, which influences thetherefore, we believe that the cutting characteristics ofdesign of the access cavity and the choice of root canal

CM wire NiTi files can be analyzed in this study.

restoration9,10）. Currently, based on the concept of MI

The purpose of this study was to analyze the effectEndo, TruNatomy（TN）files（Dentsply Sirona, Swit-of using CM wire NiTi files with minimally invasive

zerland）have been developed to allow accurate root

root canal preparation. Conventional NiTi wire PT files

canal formation without the need for SL access. How-

and CM wire PTG and PTU files were classified

ever, all other NiTi files require root canal preparationaccording to cutting characteristics, with and without

based on SL access.

SL access. The root canal morphology was compared,

The alloy materials of NiTi files have been improvedby introducing the technology of thermo-mechanicaland the suitability of CM wire NiTi files for minimallyinvasive cutting was evaluated.

treatment and a final surface finishing process. Thesematerials（M wire and CM wire）possess excellent

Materials and Methods

flexibility and fatigue resistance, but SL access is still
recommended as the standard root canal preparation

1 ．Operator and test root canal model

method. The crystal structure of NiTi alloys consists of

The operator was a dentist with more than 10 years

three phases: the austenite phase, the martensite phase,of experience using NiTi rotary files. For the experi-

and the R-layer（an intermediate phase between austen-ment, a total of 60 J-shaped root canal models（J-type

ite and martensite）, the relative proportions of whichroot canal model: curvature 30°, apical diameter #15,

determine the metallic properties. The martensite

root canal taper 02, root canal length 16.5 mm: Endphase has a monoclinic structure suitable for the prepa-

Training block canals, Dentsply Sirona）were used.

Dec, 2023

Shaping Ability of CM Wire NiTi by MI Endo
Table 1

37

The files used in root canal preparation in each NiTi file system

Group

Ni-Ti File

Orifice Opener

Glide Pass

Preparation 1

Preparation 2

Preparation 3

A
B
C
D
E
F

PTU

PTU SXPTU Slider

PTU Shaper

PTU F1PTU F2

PTU
PTG
PTG
PT
PT

O. Modifer
PTG SX
O. ModiferPT SX
O. Modifer

PTU Slider
PTU Slider
PTU Slider
PTU Slider
PTU Slider

PTU Shaper
PTG S1PTG S1PT S1PT S1

PTU F1PTG S2PTG S2PT S2PT S2

PTU F2PTG F1PTG F1PT F1PT F1

Preparation 4

PTG F2PTG F2
PT F2
PT F2
n＝10

2 ．Root canal preparation

plete root canal preparation with the same file configu-

To analyze the effect of CM wire NiTi files on theration. Before starting root canal preparation in each

final root canal morphology during minimally invasive

experimental group（A‒D）and control group（E, F）,

cutting（no-straight line（SL）access）, PTG and PTU

apical foramen penetration was confirmed by hand with

were used in the experimental groups, and PT（conven-a #10 K file, and the root canal orifice was opened intional NiTi）was used in the control group.

groups A（PTU）, C（PTG）, and E（PT）, in which SL

Root canal preparation was evaluated by comparing

access was achieved using the PTUSX, and in groups B

the displacement of the root canal width and the（PTU）, D（PTG）, and F（PT）, in which no-SL accessmedian displacement of the final root canal morphology.

used an O. Modifer. Then, the glide path of all groups

The experimental PTU and PTG groups and the con-

was formed by the PTU Slider（Dentsply Sirona）.

trol PT group were further classified into two sub-Root canals were prepared with PTU and PTG in thegroups, SL access groups and no-SL access groups,

experimental group, and PT in the control group using

making a total of six groups for analysis.

the X Smart Plus drive motor（Dentsply Sirona）. The

In each experimental group, the SL access groups

files used for root canal preparation were 3 files（PTU

used the PTU, PTG and PT files and the Orifice

Shapers, F1, F2（25/08））for PTU in groups A and B, 4Opener SX, and the no-SL access groups used an orificefiles（S1, S2, F1, F2（25/08））for PTG in groups C andmodifier as no straight line.

D, and 4 files（PT S1, S2, F1 and F2（2 5/08））for PT

The standard of the final root canal preparation filein groups E and F.

was unified for each experimental group, and the root

For each NiTi system, the file was extended to thecanal was prepared with PTU F2（file tip diameterworking length three times to shape the root canal, and#25/taper 08: ISO #25/08）and PTG F2（ISO #25/08）,

then the file was replaced. The root canal was irrigated

and the control group was similarly terminated with

with physiological saline solution every time the file

PT F2
（ISO#25/08）. Each of the PTU, PTG, and PT file

was replaced.

systems uses X Smart Plus（Dentsply Sirona）, a drive

3 ．Evaluation of root canal morphology

motor dedicated to NiTi files which rotates forward

The root canal morphological displacement was ana-

（PTU: 400 rpm/4 N cm, PTG: 300 rpm/2 to 5 N cm, PT:lyzed by comparing the amount of the root canal wall

2 5 0 r p m/ 1 t o 4 N c m）. T h e w o r k i n g l e n g t h w a s

removed at the outer and inner curved parts of thereached with three vertical movements as one cycle,

root canal. An Olympus SZX16 stereoscopic microscope

which was repeated three times, and then the file was（Olympus, Tokyo, Japan）and a DP71 digital camera

replaced. The root canal was irrigated with physiologi-（Olympus）were used to superimpose digital images ofcal saline solution after each cycle.

the transparent root canal models before and after root

Table 1 shows the files used in each experimental
and control group.
The PTU and PTG of the experimental groups andcanal preparation, and the resulting image data were
imported into a PC for measurement using WinRoof
software（Mitani Corp., Tokyo, Japan）（Fig. 1）.

the PT of the control group were analyzed by compar-

The amount of root canal wall removed was mea-

ing the NiTi file materials in a system that can com-

sured by setting measurement points located at 1, 2, 3,

ODEP Vol. 3, No. 1

38

8mm
5mm
3mm
2mm
1mm
Apex

Fig. 1 Superimposition of pre- and post-preparation images
PTU straight-line（A）, PTU no straight-line（B）, PTG straight-line（C）, PTG no straight-line（D）, PT straightline（E）, and PT no straight-line（F）.
＊：Straight-line access.

5 mm and 8 mm from the root apex, and measuring theAnalysis of the increase in the root canal width byincrease in the root canal width（inner and outer side:

PTU and PTG in the experimental group（Fig. 2）shows

distance moved from the root canal wall before root

that, regardless of the presence or absence of SL

canal preparation to the root canal wall after root canalaccess, the displacement of the outer side measurement

preparation）. Canal transportation（median root canal

was greater than that of the inner side measurement atvalues）was measured and statistically processed.

a site with a strong curvature of 1 mm. Furthermore,

4 ．Statistical processing

the outer side measurement of the PTG experimental

The measured values of root canal wall removalgroup increased more significantly than the inner side

were statistically processed using one-way analysis ofmeasurement at the measurement site of 2 mm, and novariance, the Bonferroni-Dunn multiple comparison test,

effect of SL access or no-SL access was observed.

and the Mann-Whitney U test（two-group comparison

There was no significant difference in the displacement

analysis）with a 5％ risk rate.

between the medial and lateral sides at 3‒8 mm ineither the PTU or PTG groups.

Results

In the PT control group, the outer side measurement
increased more significantly than the inner side mea-

The morphology of the root canal after root canalsurement at the 1 mm measurement site on the apicalformation was assessed at 1, 2, 3, 5 mm and 8 mm fromside, similar to the PTU and PTG experimental groups.

the root apex, and the increase in root canal width and

The displacement on the medial side increased signifi-

the median displacement on the inner and outer sides ofcantly at the 3‒5 mm measurement sites on the apicalthe root canal were measured. Figure 1 shows theside of the starting site on the inner side. There was nosuperimposed root canal models before and after root

statistically significant difference in the increase in root

canal preparation in each experimental group. Flared

canal width in the PT group, regardless of the presencemorphology of the upper third of the root canal wasor absence of SL access.

observed in groups A（PTU）, C（PTG）, and E（PT）in

There was a median displacement（canal transporta-

the SL access groups. However, in groups B（PTU）,Dtion）of 0.05‒0.1 mm on the medial side at the 3‒5 mm（PTG）, and F（PT）of the no-SL access groups, themeasurement site in the PT control group, and theflared morphology was not observed in the upper third

PTU and PTG experimental groups also showedof the root canal at the start of root canal preparation,

median displacements of 0.05 mm at the 3‒5 mm mea-

however, it was observed in the final root canal after

surement site with SL access. When comparing thethe F2 file（25/08）was used（Fig. 1）.

PTG and PTU experimental groups with the PT con-Figures 2, 3, and 4 show the increase in root canaltrol group, a significant difference was only observed inwidth and the median displacement in all experimental

the no-SL access groups. A comparison between thegroups.

PTG and PTU experimental groups showed no signifi-

Dec, 2023

Shaping Ability of CM Wire NiTi by MI Endo39

cant differences at any of the measurement sites（Fig.
3）.

8

A comparison of the median displacement between
the SL access and no-SL access groups between all4）revealed no significant difference at measurementsites from 1 to 8 mm in each group.

Discussion
NiTi files exhibit elasticity due to the coexistence ofmartensite and austenite phases, and the transformation from the austenite phase to the martensite phase is

Measurement point（mm）

NiTi files（experimental groups and control group）
（Fig.

－＊－
－＊－

5

－＊－
－＊－

3

2

initiated by external stress and temperature. The martensite phase and the austenite phase exist as temperature-dependent crystal structures, and the temperature1

at which the NiTi alloy is heated to change to the aus-

Inner side

tenite phase is defined as the Af. That is, the metallic
the crystal structure depending on the Af temperaThe PT file used in the control group for thisresearch was a conventional super-elastic NiTi wire
alloy, and because its Af is lower than the body temperature of 37℃, martensite transformation does not

Outer side

0.5 0.4 0.3 0.2 0.1 0
0 0.1 0.2 0.3 0.4 0.5
Total amount of removed canal wall
（Mean±SD（mm））
A
B
C
D
E
F

properties of NiTi alloys are determined by changes inture11‒13）.

－＊－
－＊－
－＊－
－＊－
－＊－
－＊－

Total amount of removed canal wall on the
inner and outer sides＊：Significant difference between outer and innersides（p＜0.05）.
Fig. 2

occur and it is thought that the austenite phase exists
alone. In contrast, CM wire PTG and PTU have an Afof 50‒55℃, and the alloy state is mainly composed ofthe martensite phase at the body temperature of 37℃

0.20

0.20

0.15

0.15

0.10

＊

0.05
0

1mm

2mm

3mm

（E）

（C）
5mm

（A）
8mm

－0.05
－0.10
－0.15
－0.20

Fig. 3＊

Canal transportation（mm）

Canal transportation（mm）

at the time of root canal preparation26‒29）. It is also

＊（F）

0.10
0.05
0
－0.05

＊

（D）
1mm

2mm

3mm

5mm

8mm

（B）

－0.10
－0.15
－0.20

Canal transportation at different levels from the apex in post-preparation by the PTU（A）, PTG（C）, andPT（E）straight-line group, and by the PTU（B）, PTG（D）, and PT（F）no straight-line group＊：Significant difference between experiment（PTU, PTG）and control（PT）（p＜0.05）.

ODEP Vol. 3, No. 1

40

Canal transportation（mm）

PT

PTG

PTU
0.25

0.25

0.25

0.20

0.20

0.20

0.15

0.15

0.15

0.10

0.10

（C）

0.10

0.05
0.05
（A）
（E）
0
0
0
1mm 2mm 3mm 5mm 8mm
1mm 2mm 3mm 5mm 8mm
1mm 2mm 3mm 5mm 8mm
－0.05
－0.05
（D）
－0.05
（B）
－0.10
－0.10
－0.10
0.05

－0.15

－0.15

－0.15

－0.20

－0.20

－0.20

－0.25

－0.25

－0.25

Fig. 4（F）

Canal transportation at different levels from the apex in post-preparation between SL access（by PTU（A）, PTG（C）, PT（E））and no-SL access（PTU（B）, PTG（D）, PT（F））in each NiTi system

reported that the critical stress of CM wire is in the3‒5 mm measurement sites on the apical side, increas-

range of 128‒251 MPa at 37℃, although the transforma-

ing the risk of perforation of the root canal wall due totion of austenite phase is initiated in NiTi alloys bystraightening of the curved root canal. A comparison of

external stress. Because the critical stress of CM wire

the apical displacement at 3 mm between the PT con-is lower than that of conventional NiTi wire, which istrol group and the PTG and PTU experimental groups

490‒582 MPa, CM wire maintains high flexibility due torevealed no significant difference in the SL access sub-the martensite phase even during preparation of a rootgroups; however, there was a significant difference29）

canal with strong curvature . These metallic proper-

between the PTG and PTU groups. These results con-ties of CM wire are considered to be the factors thatfirm that SL access is essential for accurate root canalcontribute to the superior elasticity and fatigue resis-

formation when using a conventional PT NiTi wire. In

tance of PTG and PTU.

the control group, the lateral curvature displacement

Our analysis of the increase in root canal width using

was greater than that in the experimental group at 1, 2,

PTG and PTU made with CM wire showed that theand 8 mm, suggesting that the risk of root canal perfo-

median displacement of the root canal was within 0.12

ration on the inner side also decreased, but this differ-

mm of the external curve measurement, indicating thatence was not significant.

the risk of root canal displacement at the apex was

Currently, at the start of root canal preparation with

reduced. In the no-SL access group, the measured dis-a NiTi file, a root canal search with a manual K file isplacement was 0.1 mm, which is almost negligible. Fur-essential, yielding information about the root canal cur-thermore, there was no significant difference in thevature, stenosis, apical foramen diameter, and root

median displacement of the root canal between the SL

canal length. It is believed that this assists with accu-

access group and the no-SL access group at all mea-

rate root canal preparation30,31）. In addition, in a curved

surement positions（1‒8 mm）on the apical side. Theseroot canal, removal of the cervical dentin ridge in the

results suggest that minimally invasive endodontic

upper third of the root canal is considered to be thetherapy that minimizes the cutting of cervical dentin

most important operation in root canal preparation,

without the need for SL access is possible as a result of

forming a smooth file glide path to the apical fora-

the enhanced flexibility of PTG and PTU made withmen32,33）. SL access and glide path preparation have

CM wire.

been considered essential procedures for the safe use of

In contrast, in the PT control group, both the SL

NiTi files. However, root canal formation in MI Endo

access group and the no-SL access group showed atherapy is based on the theory of minimizing themarked increase in the medial side displacement at theremoval of the prominent dentin in the upper third ofDec, 2023

Shaping Ability of CM Wire NiTi by MI Endo41

the root canal by SL access to prevent root fracture.

Acknowledgments

This concept conflicts with conventional dental school

We thank Helen Jeays, BDSc AE, from Edanz（https://jp.edanz.com/ac）for editing a draft of this manuscript.

teaching of endodontics. MI Endo therapy aims to minimize SL access by developing an accurate glide path toguide the files7,8）.

Conflict of Interest

In this study, we used the newly developed PTU
Slider for glide path preparation. The file used was Pro-

The authors declare that they have no conflict of interest
related to this study.

Glider（Dentsply Shirona）, which consists of one file, andis an improvement on the Path File（Dentsply Maille-

Authorship Declarationfer）, which required the use of three files. The PTU

All authors contributed significantly and are in agreementwith the manuscript.

Slider adopts ProGlider’s file taper of 2‒8％ to improvethe multi-flare shape（0.16 mm（D0）, 0.26 mm（D4）, 0.44mm（D8）, 0.68 mm（D12）, and 1.00 mm（D16））. To show
1.00 mm（D16）, it was suggested that the glide path
could be formed at the same time as forming the minimal upper root canal flare. In this study, as a result ofcomparing SL access and no-SL access in PTU and
PTG made of CM wire, root canal width displacementand displacement in all measurement sites were notfound. Furthermore, no significant difference was foundin median displacement. It is considered that this result
was influenced by the appropriate flaring created in theupper part of the root canal by using the PTU Slider.
No-SL access in minimally invasive root canal preparation requires that the surgical field is reduced at thestart of root canal preparation, which limits the operation of the instruments and makes it difficult to find aclear line of sight. Inadequate follow-up of anatomicalroot canal morphology was predicted, but successful
outcomes were achieved for root canals prepared with
CM wire NiTi files and with no-SL access for root canal
preparation.

Conclusion
This study show that minimally invasive cutting ofcervical dentin enables accurate root canal formation
using CM wire NiTi files according to the MI Endo concept, which reduces the risk of root fracture. Root canal
preparation using CM wire PTG and PTU was shownto maintain the original anatomical root canal morphology even in the no-SL access groups. PTG and PTU
files were shown to be capable of preparing root canals
using minimally invasive endodontic therapy due totheir excellent flexibility and fatigue resistance.

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32）Sanz JL, Rodríguez-Lozano FJ, Llena C, Sauro S, Forner
L. Bioactivity of bioceramic materials used in the dentin-pulp complex therapy: A systematic review. Materials（Basel）2019; 27: 1015‒1037.
33）Yu YH, Kushnir L, Kohli M, Karabucak B. Comparing
the incidence of postoperative pain after root canal filling with warm vertical obturation with resin-based
sealer and sealer-based obturation with calcium silicate-based sealer: a prospective clinical trial. Clin Oral
Investig 2021 Feb 8. doi: 10.1007/s00784-021-03814-x

by ProTaper universal and ProTaper next during shap-Dec, 2023

43

Original Article

ODEP 3（1）：43～51，2023

Discoloration of Conventional Resin Composite Cements
and Self-adhesive Resin Cements
Immersed in Water for 180 Days
Ayaka HORI-ISHIKAWA, Yuka OGAWA-UZAWA, Ayako OKADA, Daichi AIZAWA, Nana SAKAEDA-NAITO,
Hiro MATSUMOTO, Kaoru OHMORI, Masao HANABUSA and Takatsugu YAMAMOTO
Department of Operative Dentistry, Tsurumi University School of Dental Medicine

Abstract
Purpose: This study aimed to evaluate the influence of 37℃ water immersion for up to 180 days on the
discoloration of conventional resin composite cements and self-adhesive resin cements.
Methods: Three conventional resin cements（Panavia V5, RelyX Ultimate, and Variolink Esthetic DC）and
three self-adhesive resin cements（SA Luting Cement, RelyX Unicem 2, and SpeedCEM Plus）were examinedin this study. Disk specimens（⌀15.0×1.0 mm）were fabricated using the cements and their colors were measured（CIELAB）. The specimens were immersed in distilled water at 37℃ for 180 days and the colors wererepeatedly measured at 1, 7, 14, 21, 28, 90, and 180 days. The color differences（ΔE）were calculated for eachmeasurement period. In addition, water sorption, water solubility, and surface free energy of the cements
were measured. Data were analyzed using the Shapiro-Wilk test, Friedman’s test and Bonferroni corrections,and Mann-Whitney U-test.
Results: All the cements except SA Luting Cement revealed clinically acceptable values of ΔE（＜3.3）at
90 days of immersion. ΔE of the cements remarkably increased after 90 days and showed values muchgreater than 3.3 at 180 days. All the self-adhesive resin cements demonstrated significantly greater values ofwater sorption than the corresponding conventional resin composite cements. As for water solubility, one of
the three comparison pairs（Panavia V5 and SA Luting Cement）did not show a significant difference. The
surface free energy was similar among the cements at approximately 50 mJ/m2.
Conclusions: Although the water sorption of the resin cements increased due to the presence of the adhesive monomers, the discoloration did not intensify as water absorption increased. The degree of discoloration
was not related to the degree of water sorption.
Key words: self-adhesive resin cement, discoloration, ΔE＊ab, water sorption

Corresponding author: Ayako OKADA, Department of Operative Dentistry, Tsurumi University School of Dental Medicine,2‒1‒3, Tsurumi, Tsurumi-ku, Yokohama 230‒8501, Japan
TEL: ＋81‒45‒580‒8550, FAX: ＋81‒45‒573‒9599, E-mail: okada-a@tsurumi-u.ac.jp
Received for Publication: June 9, 2023/Accepted for Publication: September 3, 2023
DOI: 10.11471/odep.2023‒005

ODEP Vol. 3, No. 1

44

may be more susceptible to discoloration due to more

Introduction

water sorption; however, there is little information
available about the discoloration of resin cements. ThisThe growing demand for esthetic dentistry has

study aimed to evaluate the effects of immersion inpushed many manufacturers to develop tooth-coloredwater at a temperature of 37℃ on the discoloration ofrestorative dental materials, such as dental ceramics.

self-adhesive resin cements and conventional resin com-

Resin cements have contributed to the increased use of

posite cements. The null hypothesis was that there

ceramics for the following reasons: the bond strength ofwould be no significant differences in discoloration

resin cements allows sufficient marginal sealing of den-among the tested cements after they had been

tal restorations and reinforces ceramic restorations1,2）,

immersed in water at 37℃ for 180 days.

and the colors of these cements closely match that of
the tooth substrate.

Materials and Methods

Silica-based ceramics have sufficient translucency
and allow for the passage and scattering of light3）. The

1 ．Materials

resulting color of the restoration is dependent on the

Table 1 lists the six resin cements evaluated in thiscombination of restorative materials employed, the tar-study and their basic formulations. They included three

get tooth, and the resin cement used4,5）. Namely, theconventional resin composite cements（amine-free

discoloration of resin cement is essential for the long-dual-activated cement）: Panavia V5（PF, Kuraray Nori-term success of esthetic dental restoration. Several

take Dental, Tokyo, Japan）, RelyX Ultimate（RUl, 3M

studies have examined the discoloration of resin

ESPE, St. Paul, MN, USA）, and Variolink Esthetic DC

cements6‒11）, many of them focusing on water absorp-（VE, Ivoclar Vivadent, Schaan, Liechtenstein）, andtion as one of the key elements affecting color discolor-

three self-adhesive resin cements: SA Luting Cement

ation. The resin cement around a restoration’s margins（SA, Kuraray Noritake Dental）, RelyX Unicem 2（RUn,

is exposed to the oral environment, absorbing water

3M ESPE）, and SpeedCEM Plus（SC, Ivoclar Vivadent）.

and other substances, such as saliva, food components,

2 ．Discoloration

and beverages. This can influence the degradation andEach resin cement was mixed using mixing tips anddiscoloration of dental materials. One study reported

packed in a stainless-steel mold（15 mm in diameter, 1

the changes in color（ΔE＊ab values）exhibited by four

mm in thickness）on a glass plate（n＝5）. The resindual-cured resin cements and two chemically cured

cement was covered with a transparent plastic matrix

resin cements after one week of coffee immersion12）.

and a glass plate（thickness: 5.76 mm）. The top and

The products that demonstrated greater water sorption

bottom surfaces of the resin cement were divided intoand solubility displayed greater color changes. It wasnine zones, and each zone was irradiated using an LED

suggested that the adsorbed solution caused discolor-

curing light（PenCure 2000, Morita, Osaka, Japan）atation of the resin matrix itself, altering the resin matrix

1,048 mW/cm 2 . The light irradiation time was asat the interface of the matrix and fillers.

instructed by the manufacturer. The tip of the light

In the decade since self-adhesive resin cement was

guide was placed in contact with the glass plate. Fol-

introduced into dentistry, it has rapidly gained popular-

lowing ultrasonic cleaning for 1 min, the colors

ity due to its ease of use. Self-adhesive resin cements（CIELAB system）of the disks were measured at theare based on filled polymers, which are designed tocenter of the disks using a spectrophotometer（SE-2000,

adhere to tooth structures without needing a separate

Nippon Denshoku Industries, Saitama, Japan）on a neu-

adhesive or etchant13）. Self-adhesive resin cement con-tral gray background. Each disk was put together in atains a high concentration of adhesive monomers, suchnon-woven pouch. The specimens were immersed inas methacryloyloxydecyl dihydrogen phosphate（MDP）

distilled water（Direct Q3UV, Merck-Millipore, Darm-and glycerol phosphate dimethacrylate. As self-adhesive

stadt, Germany）
at 37℃ for up to 180 days. The distilled

resin cements contain more hydrophilic monomers thanwater was changed every week. At 1, 7, 14, 21, 28, 90,conventional resin composite cements, to give themand 180 days of immersion, the disks were ultrasoni-adhesive characteristics, self-adhesive resin cements

cally cleaned for 15 s, and their color was measured

Dec, 2023

Discoloration of Resin Cements during 180‒day Water Immersion
Compositions of the resin cements used in this study

Table 1
Product

Manufac-

name

turer

Panavia V5

45

Shade

Kuraray
Noritake Clear
DentalCodeFiller（vol％）

PF

Component

Lot No38

Paste A：Bis-GMA, TEGDMA, hydrophobic aromatic dimethacrylate, hydrophilicaliphatic dimethacrylate
Paste B：Bis-GMA, hydrophobic aromatic
dimethacrylate, hydrophilic aliphatic AX0022
dimethacrylate
Fillers：dilanated barium glass filler, silanated fluoroalminosilicate glass filler, colloidal silica, silanated alminium oxide filler

Conventional
resin composite cements
RelyX
Ultimate

3M ESPE

Translucent

RUl

43

Base：methacrylate monomers containing
phosphoric acid groups, methacrylate monomers
4801246
Catalyst：methacrylate monomers
Fillers：silanated fillers, alkaline（basic）
fillers

Variolink
Esthetic DC

IvoclarVivadent

Neutral

VE

38

UDMA and further methacrylate monomers, ytterbium trifluoride mixed oxide,
initiators, stabilizers and pigments

40

P a s t e A：M D P , B i s - G M A , T E G D M A ,HEMA, other methacrylic monomer, surface treated barium glass filler, surface
treated silica filler, light initiator, dark cure
initiator, pigment
2S0001
Paste B：surface treated barium glass filler,
aluminum oxide, surface treated sodium fluoride, polymerization accelerator, silane
coupling agent, pigment

43

Base paste：phosphoric acid-modified methacrylate monomers, bifunctional methacrylate
4748225
Catalyst paste：methacrylate monomers
Fillers：alkaline（basic）fillers, silanatedfillers

45

UDMA, TEGDMA, PEGDMA, methacrylated phosphoric acid ester, 1,10-decandiol
dimethacrylate, copolymers, dibenzoyl per- Y02748
oxide, ytterbium trifluoride, barium glass,silicon dioxide

SA Luting
CementKuraray
TransNoritake
lucentDentalSA

Self-adhesive
resin cements
RelyX
Unicem 2

SpeedCEM
Plus

3M ESPE

IvoclarVivadent

Translucent

RUn

TransSClucent

W04495using the same method as described above. Differences

3 ．Water sorption and water solubility

in color parameters（ΔL＊, Δa＊, and Δb＊）were deter-

The water sorption（Wsp）and water solubility（Wsl）

mined by comparing the values seen before and afterof the resin cements were measured as per ISO 4049.

immersion, and overall changes in color（ΔE＊ab）were

Disk-shaped specimens were prepared using the same

calculated using the following equation:

method as for the color measurement（n＝5）. Thickness

＊

＊ 2

＊ 2

＊ 2 1/2

ΔE ab＝［（ΔL ）＋（Δa ）＋（Δb ）］ …（1）was measured to an accuracy of 0.01 mm when calcu-

ODEP Vol. 3, No. 1

46

lating the volume（V）of the disks. The specimens were

5 ．Statistical analyses

kept in a desiccator at 37℃ for 22 hours. Then, they

The distribution of each continuous variable waswere placed in a second desiccator at 23℃ for 2 hours,

checked for normality using the Shapiro-Wilk test. The

before being weighed（HR-251AZ, A & D, Tokyo,

Levene test was used to assess the homogeneity ofJapan）. These procedures were repeated until a con-variances when normality was confirmed.

stant weight（m1）was obtained. It took 18 days to reachFor each cement, Friedman’s test was used to evalu-

a constant weight. The disks were then immersed in

ate whether the immersion procedure affected ΔE＊ab.

distilled water at 37℃ for 1 week. Excess water was

In addition, the comparisons among the immersion peri-

removed by blotting, and the disks were weighed（m2）.

ods for each cement were subjected to Bonferroni cor-

The specimens were stored again in the 37℃ desicca-

rection. The Mann-Whitney U-test was used for com-

tor and weighed repeatedly, as described earlier, until aparisons of Wsp, Wsl, and surface free energy between

constant weight（m3）was obtained. The following equa-

the conventional resin composite cements and the corresponding self-adhesive resin cements. The signifi-

tions were used for calculating Wsp and Wsl:

cance level was set at α＝0.05 for all statistical tests.

Wsp＝（m2－m3）/V…（2）
Wsl＝（m1－m3）/V…（3）

These analyses were carried out using IBM SPSS

4 ．Surface free energy calculation

Statistics Ver. 23.0（IBM SPSS, Tokyo, Japan）and EZR,

Disk-shaped specimens（15 mm in diameter, 1 mm inwhich is a graphical user interface for R. More pre-thickness）were fabricated using the same stainlesscisely, it is a modified version of R Commander

steel mold（n＝3）. Following light-irradiation for curing,

designed to add statistical functions that are frequently

one flat surface of the disk was serially wet-polishedused in biostatistics.

with up to 2000-grit SiC paper and mirror-finished with
3-μm diamond particles. To calculate the surface free

Results

energy of the materials, contact angles（θ）were measured using a contact angle device（DMe-201, Kyowa

1 ．Discoloration

Interface Science, Saitama, Japan）. Distilled water andTable 2 lists the changes in ΔE＊ab observed during

diiodomethane（Kanto Chemical, Tokyo, Japan）were

the immersion period. During the first 90 days of theused as liquids with known surface free energy levels

immersion period, the ΔE＊ab values for all cements,

for the angle measurement. Then, 0.5μL of each liquid

except SA, remained in the clinically acceptable range,

was dropped onto the polished surface, and video

indicating that the induced color differences were indis-

images were recorded. The video images were auto-

tinguishable to the human eye（＜3.318））. ΔE＊ab mark-matically transferred to a connected computer, on

edly increased from 90 days to 180 days（＞7.9）. The

which an image analysis program（FAMAS, Kyowa

ΔE＊ab value of SA was 0.45 at one day and graduallyInterface Science）for angle measurement had been

increased to more than 3.3 at 28 days, reaching 10.91 at

installed. The surface free energy（γStotal）of each mate-

180 days. Regarding the values seen at 180 days, two of

14‒17）

rial was calculated using the following equations:

the three conventional resin composite cements（PF and

γLtotal（1＋cosθ）＝2（ γdLγdS＋ γhLγhS）…（4）
d
h
total
γS ＝γS＋γS…（5）where γLtotal is the surface free energy of the liquids, γdLVE）exhibited slightly higher ΔE＊ab values than theis the dispersion force of the liquids（distilled water: 21.8

a significant effect on ΔE＊ab（p＜0.001）. However, Bon-mN/m and diiodomethane: 49.5 mN/m）, and γhL is theferroni correction found no significant differenceshydrogen bond force of the liquids（distilled water: 51.0

among the values for the seven immersion periods.

two self-adhesive resin cements（SA and SC）. Friedman’s test revealed that the immersion procedure had

mN/m and diiodomethane: 1.3 mN/m）. The dispersion

Figure 1 shows comparisons of the ΔE＊ab values

force（γdS）and hydrogen bonding force（γhS）of the solid

seen at 180 days between pairs of resin cements pro-were obtained by substituting the liquid surface free

duced by the same company. RUl and RUn demon-energy and the contact angle（θ）into equation（4）.

strated similar values, while significant differences in

Surface free energy of the solids（γStotal）is the sum of γdS
and γhS.

ΔE＊ab were seen between PF and SA（p＜0.001）, andbetween VE and SC（p＜0.001）.

Dec, 2023

Discoloration of Resin Cements during 180‒day Water Immersion
Table 2

Days
PF, median
（IQR）

47

Changes of ΔE＊ab

1

7

14

21

28

90

180

p-value

0.56

0.75

1.51

1.56

1.56

1.59

15.08

0.000162（0.48‒0.65） （0.74‒1.11） （1.40‒1.68） （1.54‒1.81） （1.54‒1.81） （1.49‒1.74） （4.48‒15.22）

RUl, median
0.83
0.58
1.68
1.39
1.01
1.50
12.12
0.0000715（IQR）（0.71‒0.86） （0.43‒0.69） （1.46‒1.80） （1.37‒1.42） （0.96‒1.28） （1.47‒1.54） （12.06‒12.99）
VE, median
（IQR）

0.53
0.76
1.77
1.55
1.83
2.16
13.49
0.0000415（0.48‒055） （0.75‒0.81） （1.77‒1.78） （1.51‒1.65） （1.82‒1.88） （2.15‒2.20） （13.42‒16.50）

SA, median
（IQR）

0.45
2.69
2.63
2.72
3.44
5.59
10.91
0.0000474（0.33‒0.49） （2.63‒2.70） （2.58‒2.81） （2.71‒2.94） （3.31‒3.62） （5.31‒5.68） （10.88‒10.93）

RUn, median
0.45
0.72
0.94
1.07
0.91
1.15
12.55
0.000562（IQR）（0.44‒0.47） （0.46‒0.76） （0.82‒1.00） （0.96‒1.09） （0.86‒1.29） （1.08‒1.25） （12.12‒13.46）
SC, median
（IQR）

2.82
1.98
1.04
0.93
1.19
1.89
7.91
（2.79‒2.89） （1.95‒2.01） （1.02‒1.07） （0.87‒1.08） （1.03‒1.29） （1.81‒1.93） （7.78‒8.06）

0.0000457

p‒values were calculated by Friedman’s test. There were no significant differences by Bonferroni corrections.
＊

16.00

16.00
14.00
ΔE＊ab

ΔE＊ab

14.00
12.00
10.00

12.00
10.00

8.00
PF
SA
Resin cements
a：ΔE＊ab of PF and SA

8.00
RUl
RUn
Resin cements
b：ΔE＊ab of RUl and RUn

＊

16.00

ΔE＊ab

14.00
12.00
10.00
8.00
VE

SC

Resin cements
c：ΔE＊ab of VE and SC
Fig. 1
ΔE＊ab values of self-adhesive resin cement and conventional resin cement made by the same company at 180 days

2 ．Water sorption and water solubility

manufactured by the same company. Significant differ-

Table 3 summarizes the results of comparisons ofences in Wsp were observed for all combinations of con-water sorption（Wsp）and water solubility（Wsl）between
pairs of conventional and self-adhesive resin cements

ventional and self-adhesive resin cements（p＜0.05）. In
addition, two of the pairs（RUl and RUn, and VE and SC,

ODEP Vol. 3, No. 1

48
Table 3

Water sorption（Wsp）and water solubility（Wsl）of the resin cements（μg/mm3）

PF
Wsp, median（IQR）
Wsl, median（IQR）

SA

19.67（19.62‒19.83）＊
0.50（0.00‒0.96）

RUlp‒value

28.22（28.10‒28.73） 0.00794
1.01（0.50‒1.02）＊

0.151

RUn

p‒valueWsp, median（IQR）

24.04（24.02‒25.04） 27.06（26.90‒27.59） 0.0159
Wsl, median（IQR） －3.58（－3.61‒－3.43） 3.04（2.57‒3.07）
0.00794
VE

Wsp, median（IQR）
Wsl, median（IQR）

SC

22.12（21.84‒22.13）
1.51（1.11‒1.84）

p‒value

30.88（30.78‒30.99） 0.00794
0.98（0.50‒0.99）

0.00794＊：Values having an asterisk were not significantly different by the Mann‒
Whitney U‒test（p＞0.05）.
Surface free energy（γStotal）of the resin
cements（mJ/m2）

than that of indirect restorations performed with con-

Surface free energyp‒value

research on self-adhesive resin cements has focused on

49.32（48.97‒50.90）
51.40（51.27‒52.17）

0.400

RUl, median（IQR） 52.46（51.74‒53.33）
RUn, median（IQR） 48.28（47.95‒48.65）

0.100

VE, median（IQR）
SC, median（IQR）

0.100

Table 4

PF, median（IQR）
SA, median（IQR）

46.68（45.35‒46.96）
51.48（49.95‒51.84）

ventional resin cement21）. For this reason, most major
their mechanical properties, while little information isavailable about their discoloration, despite this being anessential factor for esthetic dentistry.
Several studies have reported that self-adhesive resin
cements generally exhibit more hydrophilic propertiesp‒values were calculated by the Mann‒Whitney U‒
test.

than conventional resin composite cements because
their functional monomers contain acidic carboxyl or
phosphate groups22‒24）. Based on these findings, there
was concern about the discoloration of such cements
due to water sorption. To address this, we evaluated

p＝0.00794）exhibited significant differences in Wsl. The

the water sorption, water solubility, surface freedifference in Wsl between PF and SA was not signifi-

energy, and discoloration of conventional resin compos-

cant.

ite cements and self-adhesive resin cements. To the

3 ．Surface free energy measurement

best of our knowledge, the present study is the first toTable 4 shows comparisons of surface free energy

compare the discoloration of three self-adhesive resin（γStotal）values between pairs of conventional and self-ad-cements with the discoloration of conventional resin

hesive resin cements manufactured by the same com-composite cements from the same manufacturers.

pany. No significant differences were observed in any of

The resultant data showed that the water sorption（Wsp）of all of the cements was below the permitted

the comparisons.

ISO standard threshold of 40μg/mm3. In a comparison

Discussion

between the conventional and self-adhesive resin

Self-adhesive resin cements have been widely usedthan the corresponding conventional resin composite

for esthetic dental restoration. The procedures for

cements（Table 3）. All of the self-adhesive resin

applying self-adhesive resin cements can be very sim-

cements examined in this study contained phosphoricple, and hence rely less on technique than those foracid monomers as functional monomers, and theirconventional resin cements19,20）. However, the dentinhydrophilicity must have been reflected in the results.

bond strength of indirect restorations performed with

Moreover, the influence of polymerization must be con-self-adhesive resin cement was reported to be lower

sidered because the degree of polymerization is a criti-

cements, the latter exhibited significantly greater Wsp

Dec, 2023

Discoloration of Resin Cements during 180‒day Water Immersion

49

cal factor for Wsp. Several studies have suggested thatin SA may have affected its ΔE＊ab values. However,acidic monomers in self-adhesive resin cements may

the manufacturer did not provide the exact chemicalchemically interfere with the amine initiator, triggering

composition of SA.

incomplete polymerization and decreasing the degree of
conversion

13,25,26）

All the samples showed yellowish discoloration with

, i.e., the acidity increases the Wsp of

the exception of PF and VE groups, and SA groupself-adhesive resin cements. To solve these problems,

exhibited the most prominent discoloration after 180

non-amine initiation systems have been employed indays of immersion. However, at 180 days, the ΔE＊absome self-adhesive resin cements, including SA andvalues of all cements exceeded 3.3（Table 2）. All of the27）

RUn, which were used in this study . These cements

cements showed significant discoloration after 90 days

have a more acid-compatible nature and improve theof immersion, by which time their ΔE＊ab values had

degree of conversion and polymerization rate, which

increased 1.8-fold（SA）to 9.5-fold（PF）. These results

are expected to lead to a reduction in Wsp. Thus, wesuggested that the discoloration of the resin composite

may conclude that the influence of acidity on Wsp was

cements mainly occurred during long-term immersion

relatively minor for SA and RUn.

（for over 3 months）. Continuous exposure to water

Several studies have reported that in resin cements

accelerated the deterioration of the mechanical proper-

the compositions of the matrix and filler play a key roleties of the cements. The adsorbed water may havein W sp . Bis-GMA demonstrated greater W sp thancaused the interface between the matrix and the filler

UDMA due to the OH groups it possesses, which trans-

to dissociate and discolored the resin matrix37）. Addi-

28‒32）.

tionally, when water permeates into the resin cement,

Such behavior of monomers could not be confirmed inchanges occur in its optical properties, including alter-

this study because SC, which contained UDMA as its

ations in light transmission properties, translucency

main monomer, exhibited the highest Wsp value.

parameters, and refractive indices38）. These alterations

port dye components into deeper surface layers

The greater the percentage filler content, the lesscould lead to the deterioration of the esthetic appear-

water penetrates the resin matrix, and the smaller Wsp

ance of the resin cements. In the present study, Fried-

and Wsl will be . Although the cements examined inman’s test revealed that the immersion period had a33）this study had similar percentage filler contents

significant effect on ΔE＊ab values; however, there were（around 40 vol％）, W sp and W sl varied among theno significant differences among the ΔE＊ab values of

cements. These results were consistent with several

the cements. The number of specimens（n＝5）may

34‒36）

studies of other resin cements

explain the inconsistency between these results. Inter-.

SC demonstrated the highest ΔE＊ab value after one

estingly, the ΔE＊ab values of the self-adhesive resin

day of immersion（2.82; Table 2）. The ΔE＊ab value of

cements other than the 3M ESPE products（Figure 1b）

SA markedly increased during the first 7 days ofwere significantly lower than those of the correspond-

immersion, reaching 2.69. At 28 days, all of the cements,

ing conventional resin composite cements at 180 days

except SA, demonstrated similar values, ranging from

（Figure 1）, while there was no significant difference in

0.91（RUn）to 1.83（VE）, and only the ΔE＊ab value ofΔE＊ab between RUl and RUn since their components

SA exceeded 3.3, which suggested that the color change

were similar（Table 1）. Thus, the null hypothesis was

exhibited by SA was distinguishable to the humanrejected. The results of this study suggested that the＊eye . At 90 days, the difference between the ΔE ab

water absorption at the matrix/filler interfaces wasvalue of SA and the other cements was even moresuspected to affect the discoloration of resin cement

prominent, with the other cements continuing tomore than the hydrophilicity of the adhesive mono-demonstrate ΔE＊ab values of ＜3.3. Two of the three

mers.

18）

self-adhesive resin cements, but none of the conven-

The surface characteristics of the cements were

tional resin composite cements, demonstrated obvious

investigated by comparing the cements’ surface freechanges in ΔE＊ab, suggesting that the hydrophilicity ofenergy levels, which were calculated from their contact

the adhesive monomer in SA may have affected its

angles. The cements had similar surface free energy＊

ΔE ab values during short-term（＜3 months）storage.

levels of around 45 mJ/m2, which disagreed with the

In addition, the amount of functional monomers present

results regarding the discoloration seen at 180 days.

ODEP Vol. 3, No. 1

50This demonstrated that surface free energy and discoloration were not clearly related in this study.
The discoloration seen in this study was considered
to have occurred in the body of the cements, rather
than superficially. The storage medium used in thisstudy was deionized water, which did not contain any
pigments that could cause superficial stains on the
cements. A previous study examined the color stabilityof eight self-adhesive resin cements using four kinds of
media（distilled water, red wine, curry solution, andcress solution36））. Among the media, red wine demonstrated the highest discoloration rate, followed by the
curry solution. Distilled water revealed the lowest rate.
The interface between fillers and the matrix serves as apath for water diffusion, along which the dye components of red wine could ooze into a deeper surface
layer. The pH level varied among the four media. The
varying pH levels of the storage medium could potentially have an adverse impact on the surface structure
and roughness of the materials. Surface roughness
induced by acidic pH levels enhances the vulnerability
to external and internal discolorations of resin restorations. Further in vitro tests need to be performed
using several media.

Conclusion
Resin cements were discolored by immersion inwater, and the discoloration moderately accelerated
after 90 days. Although the water sorption of the resin
cements was increased by the presence of adhesivemonomers, discoloration was not intensified byincreased water sorption. Thus, the degree of discoloration was not related to the degree of water sorption.
Conflicts of Interests
The authors declare no conflict of interest related to thispaper.

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ODEP Vol. 3, No. 1

52

Original Article

ODEP 3（1）：52～58，2023

Comparison of Shaping Ability between Nickel-titanium Hand Filesand Nickel-titanium Rotary Files during Glide path
Masashi YAMADA, Norio KASAHARA1, Satoru MATSUNAGA2, Tomoyuki INOSE, Hiroki IWASAWA,
Ryo NAKAJIMA, Yoshiki TAMIYA and Masahiro FURUSAWA
Department of Endodontics, Tokyo Dental College
1

Department of Histology & Developmental Biology, Tokyo Dental College
2

Department of Anatomy, Tokyo Dental College

Abstract
Purpose: Glide path preparation is one use of nickel-titanium（Ni-Ti）rotary files, but few studies haveaddressed the effectiveness of Ni-Ti hand files. In this study, we compared glide path preparation abilities
between a Ni-Ti hand file and a Ni-Ti rotary file.
Methods: One board-certified endodontist conducted glide path preparation in transparent root canal models. Observations of the state of root canal preparation, centering performance, and time taken to complete
glide path preparation were compared between groups prepared using a Ni-Ti hand file（HandFlex; HF
group）and a Ni-Ti rotary file（WaveOne Gold Glider; GG group）.
Results: Although both groups maintained the original root canal morphology during enlargement, theincrease in root canal width was significantly greater in the HF group for both inner and outer sides（p＜0.05）. Centering ratio at 1 mm from the apex differed significantly between HF and GG groups（p＜0.01）.
Glide path enlargement time was significantly shorter in the GG group（p＜0.05）.
Conclusion: Ni-Ti hand files took longer than Ni-Ti rotary files to achieve glide path enlargement, butmaintained the original anatomy of the root canal in the same way.
Key words: glide path, Ni-Ti hand files, Ni-Ti rotary files, martensitic files, centering ability

Corresponding author: Masashi YAMADA, Department of Endodontics, Tokyo Dental College, 2‒9‒18, Kanda-Misakicho,
Chiyoda-ku, Tokyo 101‒0061, Japan
TEL: ＋81‒03‒6380‒9136, E-mail: myamada21@tdc.ac.jp
Received for Publication: January 24, 2023/Accepted for Publication: September 14, 2023
DOI: 10.11471/odep.2023‒006

Dec, 2023

Shaping Ability of Ni-Ti File for Glide path53

length 16.5 mm, Endo Training block canals; Dentsply
Sirona）were used in each group. These transparent

Introduction

root canal models were negotiated with a #10 stainMaintaining the anatomy of the root canal system isless-steel hand file（K-FILES, MANI, INC., Tochigi,

known to affect the success rate of root canal treat-

Japan）. Working length was set as the root canal length

ment1‒3）. Root canal preparation with a nickel-titanium

minus 1 mm. Preparation of the root canal glide path

（Ni-Ti）rotary file is effective for this purpose . Glide

was then conducted using each type of file. With thepath preparation is one of the purposes of Ni-Ti rotary

WaveOne Gold Glider, enlargement was conducted with4）

files. The glide path is the induction path for the Ni-Ti

an X-Smart Plus（Dentsply Sirona）using a reciprocatingrotary file to progress toward the root apex. The rea-motion, as recommended by the manufacturer. With thesons given for using a glide path include reducing the

HandFlex, enlargement was conducted at #15/. 04

5）stress imposed on the file and the root canal , and theusing the balanced-force technique, as recommended byenlarging effect while maintaining the anatomy of thethe manufacturer. In the GG group the file was used

6）

root canal . Stainless-steel hand files were formerly

three times in a pecking motion as a single action. In

used for glide path preparation, but rotary files canthe HF group the file was used three times in a bal-

now be used for this purpose and have been shown toance-hood-forced motion as a single action. During pathbe effective7）. In actual clinical practice, however, it ispreparation, both groups received positive-pressure irri-

often necessary to use a stainless-steel hand file, as agation with 1 mL of water from a syringe, which was

Ni-Ti rotary file that cannot be pre-curved may be

carried out after every three actions with the file ininfeasible if the root canal is inclined distally and theboth groups. Glide path preparation was terminated inpatient is unable to open their mouth sufficiently wide.

both groups when the working length was reached

The use of martensitic Ni-Ti rotary files has been（Fig. 2）. We also measured the time taken to complete

increasing in recent years. The softness of these files

the glide path in each group. After glide path prepara-means that enlargement can be carried out more safely

tion, the transparent root canal models were photo-8）

while maintaining the anatomy of the root canal , butgraphed with a stereoscopic microscope（Stemi508,

such Ni-Ti hand files are scarce. Recently developed

Axiocam ERc 5 s, Carl Zeiss, Baden-Württemberg, Ger-

Ni-Ti hand files with martensitic properties are flexiblemany）. The digital images thus obtained were imported

and can be pre-curved. The use of a Ni-Ti hand file may

into a computer, and image analysis software（Photo-

permit glide path preparation in patients for whomshop, Adobe Inc., CA, USA）was used to superimpose

Ni-Ti rotary files cannot be used. In this study, we

and measure the root canal width pre- and post-root

investigated the effectiveness of Ni-Ti hand files in glide

canal preparation. Increases in root canal width on thepath preparation in terms of root canal centering ability

inner and outer sides of the curve were measured atand time required in comparison with Ni-Ti rotary files.

points 1, 3, 5 mm and 7 mm from the root canal apex in
the root canal models9）, and centering ability was evalu-

Materials and Methods

ated by calculating the centering ratio, which was
defined as the smaller value of the inner or outer side

A HandFlex（Coltene Japan, Tokyo, Japan, HF
group）was used as the Ni-Ti hand file and a WaveOne

increase in root canal width divided by the larger
value10）（Fig. 3）.

Gold Glider（Dentsply Sirona, OK, USA, GG group）as

We then carried out statistical analyses; Student’s

the Ni-Ti rotary file（Fig. 1）. Each was used to form at-test was used to compare increases in root canalglide path in a transparent root canal model. Glide pathwidth and the preparation time, with values of p＜0.05

preparation was performed on a transparent root canalconsidered significant. The Mann-Whitney U test wasmodel by the same endodontist, who had 15 years ofused to compare the centering ratio, with values of p＜

experience.

0.01 considered significant.

As transparent root canal models, ten J-shaped epoxy
resin transparent curved root canal models（curvature30°, apical foramen diameter #10, taper 2, root canalODEP Vol. 3, No. 1

54

Fig. 1 Photograph of Ni‒Ti files used for glide path preparation
a：WaveOne Gold Glider；Ni‒Ti rotary file. The cross‒sectional form of the file is a parallelogram with multiple tapers. b：HandFlex；Ni‒Ti hand file. The cross‒sectional form of the fileis a triple‒helix cross with a 0.02 taper.

ab

Inner side

Outer side
7mm
5mm
3mm
1mm
Apex

Layering images before and after glide path
images were superimposed and the difference
between the canal configuration, in four levelsof measurement plane
a：Inner side measurement width：Location of theincreased width of the inner lateral wall of the root
canal in the measurement plane after root canalenlargement. b：Outer side measurement width：Location of the increased width of the outer lateral wall of
the root canal in the measurement plane after root
canal enlargement.
Fig. 3Fig. 2 Glide path protocols
a：Reciprocating motion and glide path protocols for
the GG group. b：Balanced‒force method and glide
path protocols for the HF group.

between the HF and GG groups, increases in width on

Results

the inner side at 5 and 7 mm from the apex were significantly greater in the HF group, and the increases in

Observations of the state of the root canal after

width on the outer side at 3 and 5 mm from the apex

preparation confirmed that the original anatomy of thewere again significantly greater in the HF group（p＜root canal had been maintained during enlargement in0.05）.

both the HF and GG groups（Fig. 4）. No obvious file
deformation or breakage occurred in either group.
Table 1 shows the results of measurements of theincrease in root canal width. In the comparison

Figure 5 shows the centering ratio. Significant differences were also evident between the HF and GG
groups at 1 mm from the apex（p＜0.01）.
Figure 6 shows the glide path preparation time.

Dec, 2023

Shaping Ability of Ni-Ti File for Glide patha : Preb : Post

55

c : Pred : Post

GG group

HF group

Root canal model before and after glide path preparation with Ni‒Ti rotary filea, b：Typical example of superimposed image for GG group. c, d：Typical example ofsuperimposed image for HF group.
The original root canal anatomy was maintained during enlargement in all groups.
Fig. 4

Table 1Comparison of the increase in root canal width diameter in each group
after glide path preparation
Inner side

Outer side

1 mm

3 mm

5 mm

7 mm

1 mm

3 mm

5 mm

7 mm

Mean

0.021

0.033

0.041a0.055b

0.021

0.029c

0.04d

0.052

SD

0.005

0.008

0.011

0.020

0.006

0.008

0.009

0.017

Mean

0.006

0.012

0.021a0.038b0.006

0.013c0.023d

0.041

SD

0.002

0.006

0.008

0.014

0.002

0.007

0.010

0.014

HF group

GG group

The same superscripted letters indicate significant differences（p＜0.05）.
The Ni‒Ti hand file showed a significantly larger increase in root canal width
diameter on the inner side.

＊

HF

GG

the HF group（147.06±74.87 s）and GG group（38.33±

1.000

Centering ratio

Mean preparation time differed significantly between
8.29 s; p＜0.05）.

0.750

Discussion

0.500

Martensitic Ni-Ti rotary files offer better cyclic
fatigue resistance and are softer than conventional aus-

0.250
0.000

tenitic files, and have been shown to permit enlarge1mm

3mm

5mm

7mm
＊: p＜0.01

Comparison of centering ratios in glide pathgroups
The closer the value is to 1, the higher the centering
ability. ＊：Significant difference（p＜0.01）
Fig. 5ment while maintaining root canal anatomy9,11）. Whenused safely, Ni-Ti rotary files are suited to preparing
the glide path while maintaining root canal morphology12,13）.
The Gold Glider is a file made of martensitic metalthat has been treated by heating and slow cooling after
cutting and processing. Although the torsional fatigue

ODEP Vol. 3, No. 1

56
HF

GG

tion performance of the HandFlex and Gold Glider

Comparison of preparation times
by glide path group(sec)using standardized resin blocks including the simulated
250

curved root canals. One disadvantage of evaluating＊

glide path preparation performance in models is thatthe softened resin adheres to the file blade during
preparation, potentially causing deformation or fractureof the instrument18）. However, the use of resin blocks

125

permitted the experiment to be conducted under standardized parameters, including working length, root
canal diameter, and angle and radius of curvature.
In this study, observations of the models showed that0both the HandFlex and Gold Glider enlarged the glide＊: p＜0.05

Comparison of preparation
times by glide path groups＊：Significant difference（p＜0.05）
Fig. 6

path while maintaining root canal anatomy. Both filedesigns are shaped as active instruments and offer highcutting efficiency19）. Root canal deviation would normally be expected to be greater on the outer side14）.
However, the inner side exhibited significantly greater
enlargement by both files, consistent with previous

resistance of martensitic metal is reportedly inferior to
14）

reports of enlargement conducted by reciprocatingthat of conventional austenitic metal , the distinctive

motions using martensitic files20）. This may have beenheat treatment given to the Gold Glider makes the filebecause of the improved tracking performance of thevery soft, which is considered to compensate for theGold Glider as a result of the reciprocating motion. A

15,16）

low torsional fracture resistance. Because a visible

similar result may have been obtained with the Hand-

titanium oxide layer forms on martensitic metals as aFlex because enlargement was conducted by the bal-

result of heat treatment, they can be given a specificanced-force technique21）, which is the origin of recipro-

surface color depending on the heat treatment tem-cating motion.

13,17）. The surface of the Gold Glider has a gold

Overall, however, the increase in root canal widthcoloration for this reason. The HandFlex is a Ni-Ti handtended to be greater with the HandFlex. In addition, afile that is also manufactured by heat treatment pro-marginally significant difference in centering ability

cessing, although the details of this processing have not

was seen for Gold Glider at 1 mm lateral to the root

been made public. However, as the surface has theapex. Root canal diameter for the model used in thissame gold-colored appearance as the Gold Glider, simi-

study was 0.01 mm, with a taper of 0.02. The tip of thelar heat treatment processing is likely involved. In

Gold Glider has a diameter of 0.15 mm with a taper ofperature

enlargement at #15/. 04, the HandFlex should therefore

0.02 up to 6 mm. Therefore, after loose filing with theprovide similar shaping abilities in glide path prepara-#10 stainless-steel hand file, the file tip prepares thetion to those of Ni-Ti rotary files. However, this has yetroot canal without resistance and with no significant

to be investigated in detail.

cutting. Preparation is then achieved at the site where

The purpose of this study was to investigate whether

the taper changes. This single glide path file produces

Ni-Ti hand files can be used without any problems inthe same effect as the crown-down technique. There-

clinical practice compared to Ni-Ti rotary files. There-

fore, we can infer that when a glide path is prepared

fore, for the comparison we selected Ni-Ti rotary files

using the Gold Glider, the root canal orifice is enlarged

that resemble Ni-Ti hand files in terms of clinical char-from near the middle of the root canal, but the apicalacteristics when used for preparing a glide path, withthird is less displaced by the cutting. On the othermovements similar to the balanced-force methods, andhand, the HandFlex has a taper of 0.04 and is larger

having similar metallic properties. For these reasons,than the Gold Glider. The diameter of the #15 Hand-

we chose the Gold Glider.

Flex is markedly larger than that of the #10 stain-

In this study, we compared the glide path prepara-less-steel hand file. Therefore, even with adequate looseDec, 2023

Shaping Ability of Ni-Ti File for Glide path57

filing, the file tip will cut from the root canal orifice totion disperses potential sources of infection12）, report-

the working length. The present results suggest that

edly leading to increased post-procedural pain and flare-

the overall amount of cutting was larger, and that slight

ups29）. Measures must be taken to deal with the large

displacement occurred at 1 mm from the root apex.

amount of cutting. Because the Gold Glider has highHowever, studies have found that differences in glide

cutting efficiency and the HandFlex cuts an evenpath systems have little effect on subsequent root canalgreater amount, both generate large amounts of cut

22,23）. Minor root canal deviation as a result

fragments. It is thus essential to pay attention to theof enlargement ≤0.3 mm at the apex has been shown

method of enlargement30）and to ensure adequate irriga-

not to pose an issue in terms of clinical outcomes24）.

tion during enlargement12,31）.

preparation

Tiny differences in the amount of cutting at the glide
path stage are thus not considered clinically significant,Conclusion

as long as no root canal deviation is evident.
The time required for enlargement and preparation

Our results indicated that the HandFlex Ni-Ti hand

was significantly shorter with the Gold Glider than withfile takes longer than the Gold Glider Ni-Ti rotary file to

the HandFlex. This was consistent with previous

prepare the glide path, but adequately maintains thereports comparing preparation time using a stain-

original anatomy of the root canal in the same way as

less-steel hand file and a Ni-Ti rotary file 16,25）. The

with the Ni-Ti rotary file.

cross-section of the Gold Glider is a parallelogram, whilethat of the HandFlex is a triple helix, with both repre-

Conflict of Interest

senting active cross-sectional forms. The Gold Glider

There are no conflicts of interest to be disclosed in thisstudy.

has two blades that are always in contact with the wall,
whereas the HandFlex has three blades, and so is considered to provide a higher cutting force into the root
canal wall. The HandFlex is thus considered to have alarger cutting volume than the Gold Glider, resulting in
inferior centering ability on the apical side. In addition,
the HandFlex has a large number of blades and theforce of these blades biting into the root canal wall isstrong. However, the HandFlex is used by hand, making rotation at a stable torque difficult. As a result, cutting time is increased. Although more time is needed
for preparation when using the Ni-Ti hand files, wehave found that it is possible to use them with little
deviation. Enlargement with the HandFlex was carriedout by the balanced-force technique, as recommended
by the manufacturer. Based on enlargement with Ni-Ti
rotary files using a pecking motion, actions were performed in sets of three. This was helpful in reducing
the stress imposed on the file23）, but given that enlargement takes longer26）there is scope for improvement inthis method of use. However, the Ni-Ti hand files canenlarge more efficiently and accurately compared to thestainless-steel hand files.27,28）
Therefore, the preparation time should not be a concern in clinical use. In addition, both of these files aretapered and generate large amounts of cut fragments.
Pushing out these fragments during root canal prepara-

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V. Comparison of apical extrusion of bacteria after glide
path preparation between manual K file, One G rotary,
and WaveOne Gold Glider reciprocation preparations.
Eur Endod J 2021; 6: 221‒225.
13）Hieawy A, Haapasalo M, Zhou H, Wang ZJ, Shen Y.
Phase transformation behavior and resistance to bending and cyclic fatigue of ProTaper Gold and ProTaper
Universal instruments. J Endod 2015; 41: 1134‒1138.
14）Gao Y, Gutmann JL, Wilkinson K, Maxwell R, Ammon D.
Evaluation of the impact of raw materials on the fatigue
and mechanical properties of ProFile Vortex rotary
instruments. J Endod 2012; 38: 398‒401.
15）Prados-Privado M, Rojo R, Ivorra C, Prados-Frutos JC.
Finite element analysis comparing WaveOne, WaveOne
Gold, Reciproc and Reciproc Blue responses with bending and torsion testsm. J Mech Behav Biomed Mater
2019; 90: 165‒172.
16）Santos CB, Simões-Carvalho M, Perez R, Vieira VTL,
Antunes HS, Cavalcante DF, De-Deus G, Silva EJNL.
Torsional fatigue resistance of R-Pilot and WaveOne
Gold Glider NiTi glide path reciprocating systems. Int
Endod J 2019; 52: 874‒879.
17）Plotino G, Grande NM, Cotti E, Testarelli L, Gambarini G.
Blue treatment enhances cyclic fatigue resistance ofvortex nickel-titanium rotary files. J Endod 2012; 40:1451‒1453.
18）Roland DD, Andelin WE, Browning DF, Hsu GH, Torabinejad M. The effect of preflaring on the rates of separation for 0.04 taper nickel-titanium rotary instruments.
J Endod 2002; 28: 543‒545.
19）Walsch H. The hybrid concept of nickel-titanium rotary
instrumentation. Dent Clin North Am 2004; 48: 183‒202.
20）Hinata G, Shigetani Y, Yoshiba K, Okiji T. Shaping ability21）Roane JB, Sabala CL, Duncanson MG Jr. The“balanced
force”concept for instrumentation of curved canals. J
Endod 1985; 11: 203‒211.
22）Alves Vde O, Bueno CE, Cunha RS, Pinheiro SL, Fontana
CE, de Martin AS. Comparison among manual instruments and PathFile and Mtwo rotary instruments tocreate a glide path in the root canal preparation of
curved canals. J Endod 2012; 38: 117‒120.
23）D’Amario M, Baldi M, Petricca R, De Angelis F, El Abed
R, D’Arcangelo C. Evaluation of a new nickel-titanium
system to create the glide path in root canal preparation of curved canals. J Endod 2013; 39: 1581‒1584.
24）Wu MK, Fan B, Wesselink PR. Leakage along apical root
fillings in curved root canals. Part I: effects of apicaltransportation on seal of root fillings. J Endod 2000; 26:210‒216.
25）Pasqualini D, Scotti N, Tamagnone L, Ellena F, Berutti E.
Hand-operated and rotary ProTaper instruments: acomparison of working time and number of rotations insimulated root canals. J Endod 2008; 34: 314‒317.
26）Hata G, Uemura M, Kato AS, Imura N, Novo NF, Toda
T. A comparison of shaping ability using ProFile, GT
file, and Flex-R endodontic instruments in simulated
canals. J Endod 2002; 28: 316‒321.
27）Carvalho LA, Bonetti I, Borges MA. A comparison ofmolar root canal preparation using stainless-steel andnickel-titanium instruments. J Endod 1999; 12: 807‒810.
28）Deplazes P, Peters O, Barbakow F. Comparing apicalpreparations of root canals shaped by nickel-titanium
rotary instruments and nickel-titanium hand instruments. J Endod 2001; 3: 196‒202.
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A, Migliaretti G, Berutti E. Postoperative pain after
manual and mechanical glide path: a randomized clinical
trial. J Endod 2012; 38: 32‒36.
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RA, Luna AS. Assessment of apically extruded debris
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Oral Pathol Oral Radiol Endod 2010; 110: 390‒394.
31）Bürklein S, Hinschitza K, Dammaschke T, Schäfer E.
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and ProTaper. Int Endod J 2012; 45: 449‒461.

Japanese）

Dec, 2023

59

Original Article

ODEP 3（1）：59～72，2023

Influence of Additional Insertion after Reaching the Working Lengthon Root Canal Transportation and Extrusion of the Gutta-percha Pointin Five Ni-Ti Rotary File Systems with Different Bending Properties
Fumiyasu MURAYAMA, Hirokazu SUGITA, Miki SEKIYA, Keisuke SAIGUSA,
Taro NISHIDA, Munehiro MAEDA and Masaru IGARASHI
Department of Endodontics, The Nippon Dental University School of Life Dentistry at Tokyo

Abstract
Purpose: The purpose of this study was to investigate the influence of additional insertion of the masterapical file（MAF）after the file has reached its working length on root canal transportation and extrusion of
the trial gutta-percha point out of the root apical foramen using five Ni-Ti rotary file systems with different
bending properties.
Methods: The cantilever bending test was used to measure the bending load of the MAF. The untreatedJ-shaped curved root canal model blocks were obtained, and the root canals were negotiated with a #10
stainless steel K-file. Root canal preparation was performed in five groups: ProTaper Gold（PTG）, TruNatomy（TN）, HyFlex EDM（HEDM）, WaveOne Gold（WOG）, and RECIPROC Blue（RCB）to around size 25
using X-smart IQ（n＝10）. The post-preparation images were then obtained and superimposed on the
pre-preparation images using image processing software. Additional insertions of MAF up to the working
length were repeated ten, twenty, and thirty times for the post-preparation block, and the images wereobtained and superimposed to measure the increase in the width diameter of the inner side and outer side.
Gutta-percha points of the same size as the MAF of each group were inserted to the position of resistance ofthe root canal wall for each block. The length to the tip of the gutta-percha point was then measured using a
digital microscope. The obtained values were statistically analyzed by the Kruskal-Wallis test with a significance level of 5％.
Results: In all files, the bending load values increased linearly and then leveled off. After unloading, the values decreased, and except for TN, the bending load became zero during unloading. At 0‒1.0 mm from theapex, transportation tended to occur on the outer side of the root canal, but at 2.0 mm and above, transportation tended to occur on the inner side. In the WOG and RCB groups, transportation to the inner side of thecanal was observed at 2.0 mm. No gutta-percha point extrusion was observed in the final enlarged model, but
the length of extrusion increased as the number of insertions increased.
Conclusion: Additional insertion of the file caused root canal transportation inward above the root canal
curvature and outward below the curvature, extending the apical reach of the gutta-percha point.
Key words: Ni-Ti rotary file, root canal transportation, extrusion of gutta-percha point

Corresponding author: Masaru IGARASHI, Department of Endodontics, The Nippon Dental University School of Life Dentistryat Tokyo, 1‒9‒20, Fujimi, Chiyoda-ku, Tokyo 102‒0071, Japan
TEL: ＋81‒3‒3261‒5698, FAX: ＋81‒3‒5216‒3718, E-mail: m-igarashi@tky.ndu.ac.jp
Received for Publication: August 3, 2023/Accepted for Publication: September 14, 2023
DOI: 10.11471/odep.2023‒008

ODEP Vol. 3, No. 1

60

reached. It has also been reported that overfilling
affects the prognosis of root canal treatment23）, since

Introduction

root canal filling is considered ideal to seal the root
Ni-Ti rotary files are available in many file systems,

canal closely up to the working length. However, nowhich differ in the properties of the Ni-Ti alloy, filestudies have reported an association between root canal

shape, usage, and preparation form1‒3）. The differencestransportation and gutta-percha point extrusion within these properties are due to the heat treatment of

the additional insertion of the MAF.

alloys such as gold wire and blue wire or the associated

The purpose of this study was to investigate thedifferences in metallic phases such as R-phase and mar-influence of additional insertion of the MAF after thetensitic phase. These files, which are flexible due to thefile has reached its working length on root canal trans-

Ni-Ti alloy, are considered to have high root canal pene-

portation and extrusion of the trial gutta-percha point

trating ability and are effective in reducing the occur-out of the root apical foramen using five Ni-Ti rotary

rence of transportation during root canal prepara-file systems with different bending properties. The

tion

4‒10）

. In recent years, efforts have been made toparameters of interest in this study were the increaseminimize the amount of root canal preparation itself

in the width diameter of the inner and outer sides, andusing file geometry, thereby reducing root fractures

the length of gutta-percha point extrusion outside thethat occur after root canal treatment 11,12）. However,root apical foramen.

even Ni-Ti rotary files, which are considered to havehigh root canal penetrating ability, have different bend-

Materials and Methods

ing properties depending on the heat treatment, metal
phase, and cross-sectional shape. For example, heat-

1 ．Ni-Ti rotar y file systems and test materials

treated files have lower bending load than those that

The file systems and gutta-percha points used in thishave not been heat-treated

13,14）, and martensitic phase

experiment are listed in Table 1. Five different 25-mm

files have lower bending load than austenitic phase

file systems were used for root canal preparation: Pro-15）

files . In addition, a parallelogram cross-sectional shape

Taper Gold（PTG group, Dentsply Sirona, Ballaigues,

has lower bending load than a triangular one16,17）.

Switzerland）; TruNatomy（TN group, Dentsply Sirona）;

In general, the end of root canal preparation when

HyFlex EDM（HEDM group, Coltène-Whaledent, Alt-using a Ni-Ti rotary file is when the tip of the filestätten, Switzerland）; WaveOne Gold（WOG group,

reaches the working length. However, few teeth have

Dentsply Sirona）; and RECIPROC Blue（RCB group,

root canals with a circular cross-sectional morphol-

VDW, Munich, Germany）. The following were used for18）

ogy . Therefore, with Ni-Ti rotary files, which are pre-glide path preparation: PTG group, ProGlider（16/.02）;

pared by rotational manipulation, contamination

TN group, TruNatomy Glider（17/.02v）; HEDM group,

remains in the root canal even after the root canal

HyFlex EDM Glidepath File（15/.03）; WOG group,

preparation is completed19）. As a countermeasure, cir-

WaveOne Gold Glider（15/.02）; and RCB group, R-Pilot

20）

cumferential filing is repeated with the same file . In

（12.5/.04）. The files used for root canal preparation androot canal preparation, file flexibility is one of the mostthe size and taper of each file were ProTaper Gold S1

important factors in maintaining the original root canal（18/.02）, S2（20/.04）, F1（20/.07）, and F2（25/.08）in

morphology15）. When stainless steel files are used for

the PTG group, TruNatomy Prime（26/.04v）in the TN

root canal preparation, they tend to cause transpor-group, HyFlex EDM OneFile（25/.08‒04）in the HEDM

tation at the root apex because of their increased

group, WaveOne Gold Primary（25/.07）in the WOG

stiffness and decreased flexibility as their sizegroup, and RECIPROC Blue（25/.08）in the RCB group.

increases

21,22）. Ni-Ti rotary files are considered to havehigh root canal penetrating ability, as well as the ability

Fifty J-shaped curved root canal model blocks（rootcanal length: 16 mm, Endo Training Bloc J-Shape ⌀15;

to suppress the occurrence of transportation. However,Dentsply Sirona）
were used as the test material for root

few studies have examined whether transportation

canal preparation. Gutta-percha points used for root

occurs when root canal preparation is continued with-canal filling were ProTaper Gold Conform Fit for theout changing the file size after the working length is

PTG group, TruNatomy Conform Fit for the TN group,Dec, 2023

Influence of Additional Insertion after Reaching the Working Length
Table 1
ProTaper Gold

Materials used in this study

TruNatomy

（PTG）

61

（TN）

HyFlex EDM（HEDM）

WaveOne Gold（WOG）

RECIPROC Blue
（RCB）

Glide path preparation（Size/Taper）［Lot No.］

ProGlider（16/.02）［1693705］

Root canal preparation（Size/Taper）［Lot No.］

ProTaper Gold TruNatomy Prime HyFlex EDM
S1（18/.02）（26/.04v）
OneFile［1550304］［1557969］（25/.08‒04）
S2（20/.04）［K88436］［1637282］
F1（20/.07）［1578319］
F2（25/.08）［1553640］

WaveOne Gold RECIPROC Blue
Primary（25/.08）（25/.07）［340726］［1638086］

Types of instrumentation

Continuous
rotary motion

Continuous rotary Continuous rotary
motionmotionReciprocating R e c i p r o c a t i n gmotionmotionSingle fileSingle file

Difference in the number of Multiple files
pieces used for root canal
preparation Single file orMultiple files

TruNatomy Glider HyFlex EDM Glide- WaveOne Gold R-Pilot（17/.02v）
path File
Glider
（12.5/.04）［1634717］（15/.03）（15/.02）［340803］［K57846］［1639239］

Single fileSingle file

Gutta-percha point［Lot No.］

ProTaper Gold TruNatomy Con- HyFlex EDM gutta- WaveOne Gold RECIPROC
Conform Fit
form Fit
percha points
Conform Fit
Gutta-Percha［0000338494］ ［0000267938］［L86806］［0000346080］ ［383575O］

Manufacturer

Dentsply Sirona

Dentsply Sirona

Coltène-Whaledent

Dentsply Sirona

VDW

HyFlex EDM gutta-percha points for the HEDM group,
WaveOne Gold Conform Fit for the WOG group, andRECIPROC Gutta-Percha for the RCB group.

Load cell

2 ．Experimental details
1 ）Cantilever bending test of Ni-Ti rotary files
To identify differences in file flexibility, a cantileverbending test was used to measure the bending load of
the MAF（Fig. 1）. The file was held 6.0 mm from the tip
coincident with the start of the block curvature, a load
was applied（load cell speed: 1.0 mm/min）until the load
point set at 2.0 mm from the tip deflected 3.0 mm, andit was then unloaded until the load cell returned to theload start point, and the load values were recorded

2.0 mm
6.0 mmFig. 1

Schematic diagram of the cantileverbending test（load cell speed：1.0
mm/min）

throughout the process（n＝10）. The load value at 2.0mm deformation was also evaluated.
2 ）Measurement of root canal transportation by
image superimposition

obtained using a scanner（GT-X970; EPSON, Tokyo,
Japan）to use as data before preparation of the curved
surface. During image acquisition, the root canal was

To clarify the amount of transportation of the root

filled with a methylene blue solution. The working

canal due to the additional insertion, a view of the cur-length was defined as the length of the root canal. Next,

vature of the root canal of the untreated blocks wasroot canal preparation was performed after checking

ODEP Vol. 3, No. 1

62
5.0 mm
4.0 mm
3.0 mm
2.0 mm
1.0 mm0 mm
A：pre

B：postD：post and ＋30

C：pre and post

Measurement points of root canal width diameter increase in this study and root canal morphology image
and superimposed image
A：pre-preparation, B：post-preparation using ProTaper Gold, C：superimposed image of pre-preparation andpost-preparation, D：superimposed image of post-preparation and thirty additional insertions
Fig. 2the negotiation of the root canal with a stainless steel

the difference between the plus and minus was used tohand K-file #10（Readysteel; Dentsply Sirona）. For root

record in which direction the root canal transportation

canal preparation, a torque control motor X-Smart IQ

was occurring.

（Dentsply Sirona）was used. File manipulation was per-3 ）Measurement of gutta-percha point length
extruded from the root apex

formed only with vertical movement along the root
canal axis and with as little brushing motion as possible.

Gutta-percha points were of the same size as the

The rotation speed and torque were in accordance with

MAF for each group. For the block with final enlarge-

each manufacturer’s instructions. The root canal wasment and additional file insertion, a gutta-percha point

filled with purified water during preparation, and thewas inserted without sealer to the position where theroot canal was rinsed with purified water every threeresistance of the root canal wall was obtained, and theor four insertions to ensure that there was no blockage

block was trial fitted into the root canal. The length ofwith a #10 K file. Final enlargement was defined as thethe gutta-percha point was then measured using a digi-time when the MAF reached the working length once.

tal microscope（VHX-7000; KEYENCE, Osaka, Japan）

The block was imaged with a scanner and superim-with the root surface as the reference position to deter-

posed on the image before the preparation was per-mine the length of extrusion of the point from the root

formed using image processing software（Adobe Photo-

apex.

shop 2022; Adobe Inc., San Jose, CA, USA）
. Next, the3 ．Statistical analysis

image after ten additional insertions of the MAF up to

The data obtained were analyzed statistically using

the working length was obtained and superimposed on

statistical software（SPSS Statistics version 28; IBM,the image at final enlargement. In addition, images

Armonk, NY, USA）with the Kruskal-Wallis test andwere also obtained for the twenty and thirty insertions

Bonferroni post hoc correction at the 5％ significanceand superimposed in the same way. The measurementlevel.

was based on the method of Yun et al.

24）, drawing aperpendicular line to the central line of the root canal

Results

and measuring the increase in canal width diameter on
the outer and inner sides of the root canal wall. The

1 ．Cantilever bending test of Ni-Ti rotar y files

measurement points in this study were 0, 1.0, 2.0, 3.0,

The results of the cantilever bending test are shown

4.0 mm and 5.0 mm from the root apex based on the top

in Fig. 3. The average bending load at 2.0 mm is shownof the root apical foramen before preparation（Fig. 2）.

in Fig. 4. For all files, the load increased linearly at the

The difference in the increase in the width diameter of

beginning of loading and remained almost flat thereaf-

the inner side from the increase in the width diameterter. It also decreased after unloading, and except forof the outer side was calculated for each file group, andTN, the bending load values became zero during

Influence of Additional Insertion after Reaching the Working Length
PTG
TN
HEDM
WOG
RCB

Bending load（N）

2.50
2.00
1.50
1.00
0.50
0.00
0.00

Fig. 3

0.50

1.00

1.50
2.00
2.50
Deformation
（mm）

3.00

3.50

Bending load-deformation curves for eachgroup

unloading.
2 ．Root canal transportation calculated by image
superimposition
File fracture due to additional insertion did not occur

63

2.50
Bending load（N）

Dec, 2023

2.00
1.50

1.93
1.61

1.00
0.50
0.00

Fig. 4

PTG

0.60

0.70

TN

HEDM

0.92

WOG

RCB

Bending load value at 2.0-mm deformation foreach grouptransportation to the inner side of the canal wasobserved at 2.0 mm.
3 ．Measurement of gutta-percha length extruded
from the root apex

in any files. Figure 5 shows the increase in diameter of

The lengths of gutta-percha points extruded from thethe outer and inner side widths when each image wasroot apical foramen at final enlargement and at ten,superimposed. Figure 5A shows an increase of about

twenty, and thirty additional file insertions from final

0.2 mm, and for the TN, the increase at 5.0 mm wasenlargement are shown in Fig. 7. Compared to theabout half the amount of the other files. Figures 5B‒D

mean values, there was no extrusion at final enlarge-

show the amount of increase for each of the ten addi-

ment in any of the groups, with values in the order oftional insertions after the preparation. The increase in

HEDM（－0.31 mm）＜WOG（－0.26 mm）＜PTG（－0.22

root canal width was about 0.02 mm for both the innermm）＜TN（－0.19 mm）＜RCB（－0.14 mm）. At ten

and outer sides after ten additional insertions, about

additional insertions, some files began to show extru-

0.06 mm after twenty additional insertions, and 0.06 mm

sion, in the order of HEDM（－0.12 mm）＜WOG（－0.08

for the inner side and 0.08 mm for the outer side aftermm）＜PTG（－0.03 mm）＜RCB（＋0.01 mm）＜TN

thirty additional insertions. Among them, the increase（＋0.07 mm）. After twenty additional insertions, extru-

of 0‒1.0 mm HEDM for the outer and 3.0‒5.0 mm PTG

sion was seen in WOG（＋0.07 mm）＜HEDM（＋0.10

for the inner were most significant. Figure 5E showsmm）＜PTG（＋0.13 mm）＜RCB＝TN（＋0.43 mm）, but

the increase before preparation and after thirty addi-

the mean value was within 0.5 mm. After thirty addi-

tional insertions, and compared to Fig. 5A, there was ational insertions, the results were WOG（＋0.20 mm）＜

tendency for transportation to occur on the outer side

HEDM（＋0.32 mm）＜PTG（＋0.57 mm）＜RCB＝TNof the canal at 0‒1.0 mm from the apex. On the other（＋0.94 mm）, with a mean value within 1.0 mm.

hand, above 2.0 mm, there was a tendency for transportation to occur toward the inner side. Figure 6 shows a

Discussion

comparison of the final enlargement of each group, theincrease in the width of the outer and inner sides forThis study was conducted to compare the amount of

the number of insertions, and the direction of transpor-

file system preparation more clearly by using a ready-tation. In the comparison of each file, there was an

made clear curved root canal model block of uniform

increase in the outer side of the canal at 0‒1.0 mm frommaterial and identical morphology.

the apex and the inner side of the canal at 3.0‒5.0 mm

In this study, bending load was first measured by afrom the apex, especially significantly at twenty andcantilever bending test. The shape of the root canal of

thirty additional insertions. In contrast, there was a

the block used in this study was almost identical to theslight increase at 0‒1.0 mm in the inner side and 3.0‒5.0

shape of the root canal displaced 2.0 mm when load was

mm in the outer side. In the WOG and RCB groups,

applied to the 2.0-mm portion from the file tip. There-

ODEP Vol. 3, No. 1

64
file
＊
＊＊

＊

＊
＊＊

4.0 mm＊ ＊＊1.0 mm＊＊＊

0.0 mm

0.10

I

＊

0.20

＊

＊

＊

＊

2.0 mm

＊
＊＊

＊＊ ＊

＊
＊
＊＊

＊＊

3.0 mm

0.00

＊
＊

0.30
（mm）

RCB
WOG
HEDM
TN
PTG

＊＊＊

＊

＊
＊＊＊

5.0 mm

0.00

0.10

0.20

O

A：pre and post

0.30（mm）

file
5.0 mm＊＊＊＊

4.0 mm

＊

＊

＊

＊

＊

0.06

I

0.04

＊ ＊
＊

2.0 mm＊

1.0 mm

0.0 mm＊0.08

3.0 mm

＊
＊

＊

＊＊

＊

＊

0.10
（mm）

RCB
WOG
HEDM
TN
PTG

＊

0.00

0.02

＊

＊

＊

0.02

0.00

0.04

O

B：post and ＋10

0.06

0.08

0.10
（mm）

＊
＊＊

file
＊

＊

＊

＊

＊

＊

＊

＊

＊
＊

＊

＊

＊
＊＊

0.04

0.02

0.00

0.00

＊

＊

＊

＊

＊

＊＊

0.02

0.04

C：post and ＋20

＊
＊＊

I

0.0 mm＊＊

1.0 mm

＊
＊
＊
＊

0.06

＊
＊ ＊

2.0 mm

＊＊0.08＊ 4.0 mm

3.0 mm

＊

0.10
（mm）

RCB
WOG
HEDM
TN
PTG

＊

＊＊
＊＊

＊

5.0 mm

O

0.06

0.08

0.10
（mm）

Comparison between files of the increase in outer diameter and inner width diameter of theroot canal and root canal transportation by the number of insertions
A：superimposition of pre- and post-preparation, B：superimposition of post-preparation and ten
additional insertions, C：superimposition of post-preparation and twenty additional insertions, D：
superimposition of post-preparation and thirty additional insertions, E：superimposition of
pre-preparation and thirty additional insertions, I：increase in diameter of the inner canal width, O：
increase in diameter of the outer canal width, ○：outlier, ☆：singular value
Kruskal-Wallis test. ＊：p＜0.05

Fig. 5

Dec, 2023

Influence of Additional Insertion after Reaching the Working Length

65

＊＊

＊＊

＊

file
5.0 mm

＊

＊
＊＊

＊

＊

＊

＊
＊

＊＊＊

0.02

1.0 mm

0.0 mm

0.00

＊＊
＊

0.04

I

＊
＊

＊ ＊

＊
＊

＊

＊

＊

＊
＊

0.06

＊ ＊
＊ ＊

2.0 mm＊＊ ＊＊＊3.0 mm

0.08

0.10
（mm）

RCB
WOG
HEDM
TN
PTG

4.0 mm

＊

＊
＊

＊

＊

0.00

0.02

0.04

D：post and ＋30

O

0.06

0.08

0.10
（mm）

＊
＊＊
＊

＊

＊
＊＊

1.0 mm

0.10

＊
＊
＊＊

＊
＊
＊

＊
＊＊

＊
＊

＊

＊
＊＊

2.0 mm

I

＊

＊ ＊
＊

＊ ＊
＊

＊
＊＊＊

0.20

RCB
WOG
HEDM
TN
PTG

＊

0.30
（mm）

＊

3.0 mm＊＊＊＊

4.0 mm＊file
5.0 mm＊

0.0 mm

0.00

0.00

0.10

E：pre and ＋30
Fig. 5

O

0.20

0.30
（mm）

Continued

fore, the bending load at 2.0 mm of the loading process

bending load of TN was low in the present study, but

was evaluated. The results showed that HEDM had thethe bending load of TN continued to be measured

second lowest bending load when loaded and the lowest

during unloading. It was considered possible that thiswhen unloaded. Therefore, HEDM exhibited high flexi-material exhibited R-phase-like dynamics with bothbility and high shape memory. PTG, RCB, and WOG

shape memory and superelastic properties, unlike

had high bending load, but the bending load became

HEDM, which exhibited similarly flexible properties.

zero during unloading. This was due to the R-phase or

In the measurement of root canal transportation inmartensitic nature of the alloys. PTG and WOG used

Experiment 2, there was a tendency for transportation

the same gold wire, but they exhibited different bend-

toward the outer side of the root canal up to 2.0 mm

16）

ing load. Hou et al.

17）

reported thatfrom the root apex. On the other hand, above 2.0 mm,

the file was more flexible when the cross-sectional

there was a tendency for transportation to occur

shape was parallelogram than when triangular. The

toward the inner side. This is thought to be due to an

cross-section of PTG was a convex triangle, and that ofinflection point at the 2.0-mm curvature of the block

WOG was a parallelogram, and the present results

used in this study, where the direction of inward andwere like theirs. Therefore, it was suggested that theoutward transportation switched. The change in trans-difference in the cross-sectional shape of the files

port direction near the curvature is consistent with theaffected the bending load. Unno et al.25） reported thatreport of Perez et al.26）. Although HEDM is considered

TN showed flexible properties due to its thin wire

to have good root canal-penetrating ability due to its

diameter of 0.8 mm, unlike other files. Similarly, thehigh shape memory at room temperature, transporta-

and Schäfer et al.

ODEP Vol. 3, No. 1

66

post＋30
post＋20
post＋10post

4.0 mm＊

3.0 mm

＊
＊

＊

stage

5.0 mm

＊

A：PTG

＊

2.0 mm＊（mm） 0.30

0.20

I

0.10

0.00

＊
＊

0.0 mm＊＊＊＊

1.0 mm

0.00

0.10

O

0.20

0.30 （mm）

5.0 mm

4.0 mm

3.0 mm

2.0 mm

1.0 mm＊＊

0.0 mm

－0.20

－0.10

0.00

Dif

0.10

0.20
（mm）

Comparison of the increase in the outer diameter and inner width diameterof the root canal and root canal transportation for each file by the number of
insertions
A：ProTaper Gold, B：TruNatomy, C：HyFlex EDM, D：WaveOne Gold, E：
RECIPROC Blue, I：increase in diameter of the inner canal width, O：increase indiameter of the outer canal width, Dif：difference between the increase in root canalwidth on the outer canal side and the increase in root canal width on the inner canalside, post：superimposition of pre- and post-preparation, post＋10：superimposition of
pre-preparation and ten additional insertions from post-preparation, post＋20：superimposition of pre-preparation and twenty additional insertions from post-preparation,
post＋30：superimposition of pre-preparation and thirty additional insertions frompost-preparation, ○：outlier, ☆：singular value
Kruskal-Wallis test. ＊：p＜0.05
Fig. 6tion to the outer wall was observed near the root apex

blades facing the opposite direction of those with con-at thirty additional insertions（Fig. 5D）. The higher

tinuous rotation, cutting is performed in counterclock-cutting efficiency of HEDM, which is fabricated by elec-

wise rotation, and torque is released in clockwise rota-

11）

trical discharge machining（EDM） , may have caused

tion to prevent biting into the root canal wall. The con-more outward transportation than other files. In addi-

tinuously rotating file is not released from the torque,tion, transportation to the inner side was observed forso outward force is applied at the curvature27）. How-

WOG and RCB at 2.0 mm（Fig. 6D, E）. This may be

ever, for files in reciprocating motion, the outward force

related to the fact that the rotation method is recipro-

generated by continuous rotation is reset by the releasecating motion. Files with reciprocating motion haveof torque. Therefore, it is conceivable that a relativeDec, 2023

Influence of Additional Insertion after Reaching the Working Length
B：TN

post＋30
post＋20
post＋10post＊＊

4.0 mm＊＊

3.0 mm＊＊＊＊

2.0 mm＊＊

1.0 mm

I

0.10

0.00

＊

0.20

0.00

0.10

＊
＊

0.0 mm（mm） 0.30

stage

＊
＊
＊
＊

5.0 mm

67

0.20

O

0.30 （mm）

5.0 mm

4.0 mm

3.0 mm

2.0 mm

1.0 mm

0.0 mm

－0.20

－0.10

0.00

0.10

Dif

C：HEDM

0.20
（mm）
stage

5.0 mmpost＋30
post＋20
post＋10post＊

4.0 mm

3.0 mm

2.0 mm（mm） 0.30

0.20

I

0.10

0.00

＊
＊
＊

0.0 mm＊＊

1.0 mm

0.00

0.10

O

0.20

5.0 mm

4.0 mm

3.0 mm

2.0 mm

1.0 mm

0.0 mm

－0.20

＊

－0.10

Fig. 6

0.00

Dif

＊

＊

0.10

Continued

＊

0.20
（mm）

0.30 （mm）

ODEP Vol. 3, No. 1

68
D：WOG

stage

5.0 mmpost＋30
post＋20
post＋10post＊

4.0 mm＊

3.0 mm＊

2.0 mm＊＊＊

1.0 mm（mm） 0.30

0.20

I

0.10

0.00

＊
＊
＊

0.0 mm

0.00

0.10

0.20

O

0.30 （mm）

5.0 mm

4.0 mm

3.0 mm

2.0 mm

1.0 mm

0.0 mm

＊
＊

－0.20

0.00

Dif

0.10

0.20
（mm）

stage

5.0 mm

＊

E：RCB

－0.10

post＋30
post＋20
post＋10post＊

4.0 mm＊＊

3.0 mm

2.0 mm＊0.20

I

5.0 mm

0.10

0.00

＊

（mm） 0.30

0.00

0.10

O

＊

0.0 mm＊＊

1.0 mm

0.20

＊
＊

＊

4.0 mm

3.0 mm

2.0 mm

1.0 mm

0.0 mm

－0.20

－0.10

Fig. 6

0.00

Dif

0.10

Continued

0.20
（mm）

0.30 （mm）

Dec, 2023＊＊

Length（mm）

1.50
1.00

Influence of Additional Insertion after Reaching the Working Lengthpared, and only the amount of two-dimensional forma-＊＊＊tion and transportation can be identified. To superim-

＊

＊
＊

＊
＊
＊

＊

pose not only one direction but the entire root canal,＊＊

the images should be obtained and superimposed in a

0.50

three-dimensional image using micro-computed tomog-

0.00

raphy imaging or other methods. This would allow for a

－0.50
－1.00

69

PTG

TN

HEDM

WOG

RCB

post＋30
post＋20
post＋10
post

Fig. 7 Distance between the apical foramen and thetip of the gutta-percha point
post：post-preparation, post＋10：ten additional
insertions from post-preparation, post＋20：twenty
additional insertions from post-preparation, post＋30：
thirty additional insertions from post-preparation,○：outlierKruskal-Wallis test. ＊：p＜0.05

three-dimensional view of the transportation of the root
canal and allow for a higher level of comparison. However, when X-rays are used, the object must be made ofa material that does not transmit X-rays, and so consideration must be given to the composition and materialsof the object.
In the present study, the file with the highest bending load was the PTG. However, transportation toward
the inner side above 3.0 mm increased with each additional insertion. Compared to the PTG, the HEDM file,which has lower bending load, showed less transportation toward the inner side. In the area near the root

inward force is applied. In other words, the file in recip-

apical foramen, transportation was greater with therocating motion has stronger contact with the inner

HEDM file, but significantly less with the PTG. Theseside of the canal than the file in continuous rotation due

results suggest that, in the area below the curvature

to the repetitive counterclockwise and clockwise rota-near the root apical foramen, transportation is related

tional motions. Therefore, transportation to the inner

to the cutting efficiency of the file used, whereas in theside at the curvature of the canal is considered to havearea above the curvature, it may be related to theincreased.

bending load of the file.

Furthermore, in all file systems, root canal straight-

In Experiment 3, additional insertion of the same size

ening was observed, with transportation to the outer

file up to the working length caused transportation inside near the root apex and transportation to the inner

the area above and below the root canal curvature,side in the upper part of the root canal. This was simi-

excessive cutting in the root apical region, and extru-

lar to that reported by al-Omari et al.28） who showedsion of the gutta-percha point out of the root apicalroot canal straightening in a stainless steel file. There-

foramen. In root canal preparation using Ni-Ti rotaryfore, even when Ni-Ti files with high root canal-pene-

files, root canal filling is usually performed with a

trating ability were used, additional insertion caused

matched-taper, single-cone technique using a gutta-per-straightening of the root canal as with stainless steel

cha point with a matching size and taper. In addition,

files.

unlike root canal preparation with a stainless steel hand

In the analysis using transparent root canal blocks, as

file, the gutta-percha point is not held in place by anin this study, a digital camera or flat head scanner isapical sheet or apical collar, but rather by the entire

29‒31）

Although the image acqui-root canal preparation surface. In the present study,

sition methods are different, it is possible to analyze

excessive additional file insertion may have resulted inchanges in root canal morphology in two dimensions byenlargement of the formation surface, thereby reducing

fixing the shooting position and setting the criteria for

its retention capacity and leading to extrusion of thesuperimposition to something other than the root canalgutta-percha point.

used to acquire images.

morphology. However, the comparison test of the

In the present study, the root apical foramen wasamount of root canal preparation and transportation bydefined as the narrowest part of the block used, and thesuperimposition of two-dimensional images is per-root canal length was set as the working length. The

formed by superimposing images from only one direc-

most constricted area in human teeth is the physiologi-tion. Therefore, only the curvature view can be com-cal apical foramen, but there is a difference of approxi-

ODEP Vol. 3, No. 1

70mately 1.0 mm between the physiological apical fora-

root canal preparation alone.

men and the anatomical apical foramen32,33）. Wu et al.34）
and Tsesis et al.35）also stated that the position of work-

Conclusion

ing length should be set between the physiological andanatomical apical foramen. Transportation may haveThis study investigated the influence of additional

destroyed the physiological apical foramen due to theinsertion of the MAF after the file has reached its

additional insertion of the file. However, the length ofworking length on root canal transportation and extru-

the gutta-percha point that was extruded in this study

sion of the trial gutta-percha point out of the root apical

was approximately 1.0 mm, suggesting that extrusion offoramen using five Ni-Ti rotary file systems with differ-

the gutta-percha point to the anatomical apical foramenent bending properties. The following conclusions were

may not occur. There was no significant difference

reached.

between the final enlargement and the block with ten

1．In the bending load measurement, the averages ofadditional insertions from the final enlargement, sug-

the load values when loading a 2.0-mm position weregesting that the tip of the gutta-percha point remained

compared, and the relationship was PTG＞RCB＞WOG

within the anatomical apical foramen. Therefore, it was＞HEDM＞TN.

suggested that up to ten additional insertions may be

2．Additional insertion of the file up to its working

acceptable if the insertion method is such that nolength caused transportation to the inner side above thebrushing motion is performed, as in the present study.

root canal curvature and to the outer side below the

However, Ricucci et al. 36） reported that root canal

curvature, with an increasing degree of transportation

preparation and filling beyond the physiological apicaland a tendency to straighten the root canal.

foramen has a poor prognosis. As a countermeasure, it

3．In the reciprocating motion file, transportation tois possible that a gutta-percha point of the same size as

the inner side occurred at the root canal curvature, andthe MAF could be used by cutting the tip of the gut-

the increase in root canal width diameter was signifi-

ta-percha point by 1.0 mm without causing extrusion

cantly greater with RCB compared to TN and HEDM

even through the physiological root apical foramen.

at the final enlargement and at the superposition of

However, even if the gutta-percha point is used with athirty additional insertions.

1.0-mm cut, the conformability of the gutta-percha point

4．Additional insertion of the file up to the working

decreases due to the occurrence of root canal transpor-

length extended the apical reach of the gutta-percha

tation and hyperenlargement if additional file insertions

point, and an average of 0.94 mm of extrusion from theare made more than ten times. As a result, a gap

root apical foramen was observed for RCB and TN

between the root canal wall and the gutta-percha point

after thirty additional insertions.

may occur. Eymirli et al.37） reported that the use of a5．Up to ten additional insertions to the working

gutta-percha point of the same size and taper as thelength of the file after the working length is reached in

MAF promoted the penetration of the bioceramicroot canal preparation may be acceptable.

sealer into the dentin tubules, leading to improved point
conformance. Therefore, if further root canal prepara-

Conflict of Interesttion is desired after considering the compatibility of the

The authors declare no conflict of interest related to thispaper.

gutta-percha point with the root canal wall, the same
size file should continue to be used, and no additional
insertions should be made. Instead, root canal preparation should be performed with the minimum number of
insertions up to the working length using a larger size
file, followed by root canal filling using a gutta-percha
point of the same size. It is also important to promote
the reduction of bacteria in the root canal by sonication
with sodium hypochlorite solution or by chemical cleaning using ultrasonic cleaning, rather than by mechanical

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Dec, 2023

73

Original Article

ODEP 3（1）：73～79，2023

Clinical Assessment of Root Caries Treatment Based on the Discolorationof Silver Diamine Fluoride as a Caries Indicator
Yuki MURASE1, Hanemi TSURUTA1, Tomohiro TAKAGAKI1, Shusuke KUSAKABE1,
Masaomi IKEDA2, Michael F BURROW3 and Toru NIKAIDO1
1

Department of Operative Dentistry, Division of Oral Functional Science and Rehabilitation,
Asahi University School of Dentistry2

Oral Prosthetic Engineering, Medical, and Dental Science and Technology, Graduate School of Medical and Dental Sciences,
Tokyo Medical and Dental University（TMDU）
3

Restorative Dental Sciences, Faculty of Dentistry, University of Hong Kong

Abstract
Purpose: This clinical study aimed to evaluate whether changes were observed in saliva test outcomes
before and after application of 38％ silver diamine fluoride（SDF）in elderly patients with root caries lesions.
Following this, the root caries discolored by SDF was restored to verify SDF as a potential caries indicator
for the removal of carious tissue on root surfaces.
Methods: This study was approved by the Research Ethics Committee of Asahi University, School of Dentistry（Approval Number: 32031）. Twenty elderly patients with root caries visiting the Operative Dentistry Clinicof Asahi University Medical and Dental Center between March 2021 to March 2022 were recruited. The caries risk of these patients was evaluated using a saliva test kit（Salivary Multi Tests; SMT）before and after
SDF application（at the first and the second visits）. The values of SMT measurement before and after SDF
application were analyzed for six items: cariogenic bacterial count, saliva acidity, buffering capacity, leukocytes, protein, and ammonia. Caries removal was guided by the staining from SDF as a caries indicator using
an excavator, then a resin composite restoration was placed. Each clinical procedure was photographed.
Results: There were 20 participants: 15 males and 5 females（mean age 76.5 years）. The total number ofteeth with root caries lesions was 90（51 incisors, 23 premolars, and 16 molars）. There were no significant
differences in the SMT results before and after the SDF application（p＞0.05）. However, the coefficient ofvariation values before and after SDF application tended to be different among the evaluated items. The
coefficient of variation was 1.97 for caries-causing bacteria, while it ranged from 0.26 to 0.69 for the otheritems. From the photographs of the clinical cases, application of SDF discolored and arrested the root caries
lesions. This led to a reduction in bacterial adherence as well as gingival inflammation. The resin composite
restorations for the teeth with root caries lesions were deemed successful during the observation period（max. 10 months）.
Conclusion: No significant difference was found in each item of SMT measurement before and after theapplication of SDF. However, the bacterial count exhibited a significant variation, suggesting suppression of
the bacterial adherence on the root caries lesion by the application of SDF. SDF application could be used asa favorable indicator for diagnosis and excavation of carious root tissue when a restoration is required.
Key words: silver diamine fluoride, root caries, saliva test
Corresponding author: Hanemi TSURUTA, Department of Operative Dentistry, Division of Oral Functional Science and Rehabilitation, Asahi University School of Dentistry, 1851, Hozumi, Mizuho, Gifu 501‒0296, Japan
TEL & FAX: ＋81‒58‒329‒1442, E-mail: hanemi@dent.asahi-u.ac.jp
Received for Publication: May 31, 2023/Accepted for Publication: October 6, 2023
J-STAGE advance published date: November 8, 2023
DOI: 10.11471/odep.2023‒007

ODEP Vol. 3, No. 1

74

noted to be an esthetic drawback13,14）. It was demonstrated that the discoloration sites after the application

Introductionof SDF to carious dentin almost coincided with the carThe 8020 movement has been promoted by the Japa-

ies-infected stained dentin（non-remineralizable tissue）

nese Ministry of Health, Labour and Welfare and thewhen a caries detector dye solution was applied15）. This

Japan Dental Association since 1989; this is a campaign

means that root dentin caries could be removed by

to preserve more than 20 natural teeth in people by theusing SDF discoloration as a caries indicator. It wasage of 80 years so as to enjoy a high quality of oral

reported that the site of root caries dentin stained red

health, which influences overall quality of life. Today,

by a caries-detecting dye solution（Caries Check, CC,

more than half of the elderly population have achieved

Nishika, Shimonoseki, Japan）almost coincided with that1）

this goal . However, there is a new concern about theof SDF, however, the site discolored with SDF wasincreased risk of root caries in the elderly population

harder than the CC-stained site, suggesting that SDF

because of root surface exposure due to recession of

application could also precipitate reminerals into theperiodontal tissue, decrease of saliva secretion, and also

caries-infected dentin15）.

a decline of patients’ manual dexterity which affects
2）

The present clinical study aimed to evaluate whether

oral hygiene . A decrease of saliva leads to a decrease

the values obtained from a saliva test before and afterin buffering capacity and also a reduced supply of Ca/

application of 38％ SDF to root caries in elderly

3）

PO4 ions which are necessary for remineralization . In

patients differed. Following this, the root caries wasaddition, an increasing number of elderly people withremoved according to the zone of discoloration（SDF-

physical/mental disabilities are unable to receive suffi-

stained zone）to verify whether SDF has the potential

cient oral care4）. Thus, attention should be paid to theto be used as a caries indicator for the removal of cari-prevention and management of the initial stages of rootous root tissue.

caries.
Root caries progresses circumferentially around theMaterials and Methods

5）

exposed root surface ; this makes the restoration ofroot caries challenging because of the difficulty of con-

1 ．Research participants

trolling moisture as well as manipulating instruments

This study was approved by the Research Ethics

during preparation and restoration due to the curvature

Committee of Asahi University, School of Dentistryof the surface. The clinical treatment of root caries（Approval Number: 32031）. The participants were

includes caries risk management and the arresting ofelderly patients requiring treatment of root caries

active root caries in its early stages. Recently, several

lesions who attended the Operative Dentistry Clinic,

studies6‒8） have reported that 38％ silver diamine fluo-

Asahi University Medical and Dental Center betweenride（SDF）is highly effective in inhibiting the progres-March 2021 and March 2022. Informed consent wassion of active root caries lesions, and its excellent inhib-

obtained from 20 patients, which included the purpose

itory effect was stated in the caries treatment guide-of this study and detailed information about the treat-lines of the Japanese Society of Conservative Den-ment of root caries including the use of SDF.

tistry9）. Thirty-eight percent SDF contains high concen-

2 ．Saliva test

trations of fluoride and silver ions. Both ions play

Saliva tests for each participant were performed

important roles in inhibiting the initiation and progres-twice: before and after SDF application（at the first andsion of root caries; fluoride ions are mainly effective forthe second visits）using a proprietary saliva test kit

the mineral component of dentin（hydroxyapatite）to（Salivary Multi Tests, SMT, Lion, Tokyo, Japan）. Data

inhibit dentin demineralization and promote remineral-

were obtained from the SMT and compared before andization10）, while silver ions are known to adsorb onto

after the application of SDF for each participant. The

11）

following six items were tested, namely cariogenic bac-and are also known to have an antibacterial effectterial count, saliva acidity, buffering capacity, leuko-

the dentin collagen to inhibit collagen degradation

12）

inhibiting bacterial adhesion onto the dentin surface .

However, discoloration of SDF-treated dentin has beencytes, protein, and ammonia.
The mean values of the test data obtained before andDec, 2023

Clinical Assessment of Root Caries Treatment with SDF Application in Elderly Patients
Table 175

Age, sex, and number of root carious teeth for each participant
Number of root caries

Sex

Subject

Age

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

68
70
70
71
72
72
72
73
75
75
75
77
79
81
81
81
83
84
85
86

M
M
F
M
M
M
M
F
M
M
M
M
M
F
F
M
M
F
M
M

Total
Mean±SD

―76.5±5.5

M15/F5―（Male/Female） Anterior tooth

Premolar

Molar

Total

0
3
1
2
1
3
2
0
1
12
4
5
1
3
3
2
1
3
1
3

1
0
2
0
0
3
0
1
0
0
0
1
1
4
0
1
2
2
2
3

0
3
1
1
1
2
0
1
0
0
0
0
0
1
0
2
0
1
1
2

1
6
4
3
2
8
2
2
1
12
4
6
2
8
3
5
3
6
4
8

51
―

23
―

16
―

90
―

after SDF application were compared and statisticallypresent. In addition, a caries-detecting solution（Caries

analyzed using a paired-sample t-test and Wilcoxon

Check）was applied to the lesion to confirm the com-signed-rank test at the 5％ level of confidence. In addi-

plete removal of the caries-infected dentin. If the lesiontion, the change rate（the ratio of the test values before

was stained red by the caries detector, those parts ofand after SDF application）was calculated for each itemthe lesion stained red were also removed. Thereafter, ato obtain a coefficient of variation（the ratio of the stan-

two-step self-etch adhesive（Clearfil SE Bond 2, Kuraraydard deviation to the mean）.

Noritake Dental, Tokyo, Japan）was applied to the cav-

3 ．Photographing of the root caries

ity and a resin composite（Clearfil Majesty ES Flow,

Each patient’s root caries lesions were photographed

Kuraray Noritake Dental）was placed according to thebefore and after the application of SDF to confirm the

manufacturer’s instructions. Photographs were taken

presence of discoloration of the root caries due to thefor each restorative step to investigate the applicability

SDF treatment. From the photographs, the presence ofof 38％ SDF for root caries treatment.

biofilm formation on the carious lesions and the degreeof gingival inflammation associated with the teeth hav-

Results

ing root caries lesions were also evaluated.
4 ．Root caries treatment

1 ．Participants and root carious teeth

For treatment of the root caries lesions at the second

Data on the age, sex, and number of teeth with root

visit, the carious lesions were removed based on black

caries lesions of each participant are summarized indiscoloration from the SDF as the indicator. A spoon

Table 1. Twenty patients（15 males and five females）

excavator（Round #2, YDM, Tokyo, Japan）was used towith a mean age of 76.5 years old were recruited. The

remove caries until the discoloration was no longer

total number of root caries lesions included in thisODEP Vol. 3, No. 1

76
Table 2

Values of SMT measurement, change rate, and coefficient of variation for each item

Inspection item

Before＊After＊

Change rate

Coefficient of variation

Cariogenic bacterial countSaliva acidity
Buffering capacity
Leucocyte
ProteinAmmonia22.9±16.6
54.2±21.7
40.1±23.1
75.7±21.8
66.3±22.747.2±22.5

34.7±25.1
54.6±21.1
42.1±24.1
73.0±25.2
66.7±22.6
43.4±27.7

10.89±21.43
1.22±0.84
1.25±0.74
1.01±0.27
1.16±0.59
1.09±0.58

1.97
0.69
0.59
0.26
0.51
0.53

＊：There were no significant differences in each item between before and after the SDF application period（p＞0.05）.

100
90
Cariogenic bacterial count3 ．Representative case of root caries treatment

Increaseusing SDF

No change

80

Figure 2 shows a representative case of the root car-

Decrease70

ies treatment using SDF. Root caries lesions werelocated on the lower incisors（#32 and #33）, as shown

60

in Fig. 2‒a. The teeth were cleaned using a brush and50prophylaxis paste（Merssage Plus, Fine, Shofu, Kyoto,

40

Japan）. SDF was then applied on the root lesions for 3

30

minutes with a micro brush（Fig. 2‒b）. The SDF-treated20

lesion showed discoloration at the second visit after one

10

week（Fig. 2‒c）. After the tooth surface was cleaned,0

BeforeAfter
SDF application

Fig. 1

Change of cariogenic bacterial count before
and after SDF application for each patient,showing an increase in 14 cases（p＜0.05）, nochange in 1 case（p＞0.05）and a decrease in 5
cases（p＜0.05）, statisticallythe SDF-stained area could be clearly observed withthe lesion showing a surface that was much harder andarrested, while the surrounding intact enamel and dentin of the teeth were not discolored. The gingival
inflammation appeared to be reduced around the SDFtreated root surfaces（Fig. 2‒d）. The black-discoloredlesions were carefully removed using a spoon excavator（Round #2, YDM）
（Fig. 2‒e）. The lesion was stained

study was 90（51 anterior teeth, 23 premolars, and 16

red by a caries detector dye solution（Caries Check）,

molars）.

which revealed that many lesions had only been partly

2 ．SMT measurement

excavated（Fig. 2‒f）. After completion of cavity prepa-

Table 2 shows the values of SMT measurement, theration（Fig. 2‒g）, a resin composite was placed in thechange rate and the coefficient of variation for each

usual manner（Fig. 2‒h）. No discomfort was observed in

item. There were no significant differences in each item

any case during the follow-up period（maximum period:

between the before and after SDF application periods

10 months）.

（p＞0.05）. The coefficient of variation was 1.97 for the
cariogenic bacterial count, while that of the other items

Discussion

ranged from 0.26 to 0.69. Figure 1 shows the change of
cariogenic bacterial count before and after SDF applica-

Root surface exposure due to recession of the peri-tion for each patient. The bacterial counts varied forodontal tissue and biofilm accumulation on the root sur-each patient, including an increase in 14 cases（p＜0.05）,

face can easily lead to the formation of root caries inno change in 1 case（p＞0.05）and a decrease in 5 cases

the elderly if all cariogenic factors exist5）. In addition,（p＜0.05）.

the risk of caries increases in the elderly due to adecrease in saliva secretion, and difficulty in self-care ofDec, 2023

Clinical Assessment of Root Caries Treatment with SDF Application in Elderly Patients
a

b

c

d

e

f

g

h

Fig. 2 Representative case of root caries treatment using SDF
a：Root caries lesions（#32 and #33）
. b：SDF was applied for 3 minutes after cleaning. c：
Discoloration of the SDF‒treated lesion after one week. d：The SDF‒stained area could beclearly observed, while the surrounding intact enamel and dentin of the teeth were not discolored. The gingival inflammation was reduced around the SDF‒treated root surfaces. e：
The black‒discolored lesions were carefully removed using a spoon excavator. f：The lesionthat was stained red with Caries Check was observed to only have been partly excavated.
g：Completion of cavity preparation. h：Final restorations.

77

ODEP Vol. 3, No. 1

78

their remaining teeth 2‒4）. Therefore, general oral

SDF based on fluorescence photomicrographic observa-

hygiene instruction is often not sufficient for managing

tions12）. This means that the biofilm formed on the root

the risk of root caries in the elderly16）. A more defini-dentin surface was easily removed from the SDF-

tive and effective approach is necessary for root caries

treated dentin surface, even if the bacteria that weremanagement and treatment for this group of patients.

left on the surface were probably inactive. In the cur-This study aimed to evaluate the effect of SDF appli-

rent study, the results of the bacterial counts were also

cation on root caries lesions as well as SMT measure-variable（Fig. 1）. The caries risk factors of the elderly

ment. The study also investigated the clinical efficacy ofpatients were variable, including saliva secretion and

SDF application for the management and treatment ofquality of oral hygiene4）. From the examination of theroot caries. Since this study was performed during thephotographs before and after SDF application 2）, the

COVID‒19 pandemic, 20 patients with 90 root carious

SDF-treated lesions were discolored and seemed

teeth could be recruited.

arrested and also gingival inflammation seemed

Saliva tests were performed using SMT to obtain thefollowing six items

17）

improved. These facts suggested that SDF application. The cariogenic bacterial countcould prevent the attachment of bacteria on the treated

was obtained from measurements of the reductive

root surfaces even if the oral hygiene of patientsactivity of resazurin with gram-positive bacteria. Values

remained poor. Further studies should be carried out tofor saliva acidity were obtained from hydrogen ion con-evaluate the effect of SDF application on the periodon-

tent detected by color change of a pH indicator, while

tal status by using periodontal evaluation measure-buffering capacity was determined by color change ofments, such as probing depth and bleeding on probing.

complex pH indicators in the presence of defined

The amount of saliva secreted by the elderly patientsamounts of acid. It is important to know the above

who participated in this study was greatly reduced,

three items since they are key indicators to determine

making saliva collection challenging. Sufficient control of

the caries risk level of patients. The values for leuko-

the intraoral environment before saliva collection was

cytes were obtained by the measurement of enzymatic

also challenging. SMT measurement was performed in

activity of leukocyte esterase using a dye-binding tech-this study, however, caries risk assessment using SMT

nology used in urinary examination. The protein values

measurement may be challenging for elderly patients.

were obtained by measuring total protein content alsousing a dye-binding technology used for urinary exam-

Conclusions

ination. The items for leukocyte and proteins are
related to determining periodontal disease. The values

1．No significant difference was found in the sevenfor ammonia were detected from an enzyme cycling

items of SMT measurement before and after the appli-

method used in blood examination, which was related tocation of SDF. However, the bacterial count exhibited ahalitosis.

significant variation, suggesting that SDF application

Interestingly, the value obtained by SMT measurement did not change for each parameter before andmay influence bacterial adhesion on dentin surfaces
affected by root caries.

after SDF application（Table 2）. However, the coeffi-

2．From the clinical cases, the application of SDF

cient of variation of the cariogenic bacteria count wasdemonstrated discoloration of the root caries lesions,1.97, while the coefficients of the other five items wereand an anti-inflammatory effect of the gingiva aroundaround 0.7 or less. This fact suggested that SDF appli-

the root carious teeth.

cation impacts only the results for the bacterial count.
Takahashi et al.12）evaluated the effect of SDF applica-

3．SMT measurement might be challenging for caries risk assessment for elderly patients.

tion on root dentin with regard to biofilm formation
using an oral biofilm reactor. They concluded that bio-Acknowledgment

film formation and insoluble glucan adhesion were pre-This study was supported by an 8020 Research Grant for
fiscal 2021 from the 8020 Promotion Foundation（21‒5‒10）
and a Grant-in-Aid from the Japan Society for the Promotionof Science（21K09924）.

vented by the application of 38％ SDF. They also
reported that almost all Streptococcus. mutans cells
were dead on the root dentin surface treated with 38％

Dec, 2023

Clinical Assessment of Root Caries Treatment with SDF Application in Elderly Patients79

Conflict of Interest

S, Kitasako Y, Kubo S, Nakashima S, Nikaido T, Shimizu

There are no conflicts of interest to disclose regarding

A, Sugai K, Takahashi R, Unemori M, Yamaki C. Evi-

this study.

References
1）Shinsho F. New strategy for better geriatric oral healthin Japan: 80/20 movement and Healthy Japan 21. Int
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KCM, Chu CH. Common medical and dental problemsof older adults: A narrative review. Geriatrics（Basel）
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A, Berardino G, Mollica A, Coelho-Júnior HJ, De Pergola
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5）Kreher D, Korn V, Meißner T, Haak R, Schmalz G, Ziebolz D. How do carious root lesions develop after theend of professional preventive measures?―Preliminary
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6）Zhang W, McGrath C, Lo EC, Li JY. Silver diamine fluoride and education to prevent and arrest root caries
among community-dwelling elders. Caries Res 2013; 47:284‒290.
7）Oliveira BH, Cunha-Cruz J, Rajendra A, Niederman R.
Controlling caries in exposed root surfaces with silver
diamine fluoride: A systematic review with meta-analysis. J Am Dent Assoc 2018; 149: 671‒679.
8）Zhang OL, Niu JY, Yin IX, Yu OY, Mei ML, Chu CH.
Bioactive materials for caries management: A literature
review. Dent J（Basel）2023; 11: 59.
9）Hayashi M, Momoi Y, Fujitani M, Fukushima M, Imazato

dence-based consensus for treating incipient enamel
caries in adults by non-invasive methods: recommendations by GRADE guideline. Jpn Dent Sci Rev 2020; 56:155‒163.
10）Shimizu M, Matsui N, Hamba H, Obayashi S, Sayed M,
Takahashi M, Tsuda Y, Nikaido T, Tagami J. Micro-CT
assessment of the effect of silver diammine fluoride on
inhibition of root dentin demineralization. Dent Mater J
2021; 40: 1041‒1048.
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Nikaido T, Tagami J. Effects of zinc fluoride on inhibiting dentin demineralization and collagen degradation invitro: A comparison of various topical fluoride agents.
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12）Takahashi M, Matin K, Matsui N, Shimizu M, Tsuda Y,
Uchinuma S, Hiraishi N, Nikaido T, Tagami J. Effects ofsilver diamine fluoride preparations on biofilm formation of Streptococcus mutans. Dent Mater J 2021; 40:
911‒917.
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ODEP Vol. 3, No. 1

80

Original Article

ODEP 3（1）：80～85，2023

The Effects of Dietary pH Changes on Tooth Staining of the Enamel Surface
Hiroki SUGITO1,2, Takako EGUCHI1, Rika TANAKA3, Akiko HARUYAMA2 and Takashi MURAMATSU2
1

Department of Dental Hygiene, Tokyo Dental Junior College

2

Department of Operative Dentistry, Cariology and Pulp Biology, Tokyo Dental College
3

Misono Garden Dental Clinic

Abstract
Purpose: Patients’ demand for dental esthetics has increased in recent years. Additionally, teeth bleaching
can improve self-care and oral cavity awareness. However, bleaching is not permanent as the consumption offoods and drinks can cause re-discoloration of teeth. Tooth staining may be caused by drinks such as coffee,
wine, and tea, and foods such as curry. However, the mechanism by which these substances cause staining
has yet to be clarified. Therefore, in this study we examined the effects of colored food on the enamel surface by dietary pH changes.
Methods: Extracted mandibular anterior bovine teeth were used as samples. The enamel samples weredivided into four groups: turmeric（TM group）, red pepper（RP group）, tomato（TO group）, and Milli-Q
water（MQ group）as a control. The samples were immersed in 30 mL of solution for 3 hours in a 37℃ incubator while being shaken. Color changes were measured before and after immersion. For colorimetry, L＊, a＊and b＊ in the CIE1976L＊a＊b＊ color system were measured using a micro-area spectrophotometer, and ΔL＊,

Δa＊, and Δb＊ were obtained. Furthermore, the immersion solution was evaluated before and after immersion using a pH meter. A 3D laser microscope was used to measure the surface roughness of enamel.
Results: The results revealed that the Δa＊ value, which indicated redness, and the Δb＊ value, which indicated yellowness, increased due to the influence of natural ingredients in the TM and RP groups. The various natural pigments in food, such as curcumin in the TM group and carotenoids in the RP group, arethought to be involved in the coloration. The enamel surface roughness of the TO group changed significantly before and after immersion. The pH of the TO group was 4.0, which was below the enamel critical pHof 5.5.
Conclusion: The results of this study suggest that tooth staining of the enamel surface is affected bydietary pH changes.
Key words: tooth staining, colorimetry, surface roughness of enamel

Corresponding author: Hiroki SUGITO, Department of Dental Hygiene, Tokyo Dental Junior College, 2‒9‒18, Kanda-Misakicho, Chiyoda-ku, Tokyo 101‒0061, Japan
TEL: ＋81‒3‒6380‒9105, FAX: ＋81‒3‒6380‒9635, E-mail: sugito@tdc.ac.jp
Received for Publication: August 18, 2023/Accepted for Publication: October 27, 2023
DOI: 10.11471/odep.2023‒009

Dec, 2023

The Effects of Dietary pH Changes on Tooth Staining of the Enamel Surface

Introduction

81

Materials and Methods

In recent years, people have become increasingly

1 ．Sample preparationinterested in esthetics and request teeth bleaching.

Forty-eight extracted mandibular anterior bovine

Teeth bleaching can improve teeth color, making peo-teeth that had been cryopreserved were used as sam-ple more aware of maintaining their teeth color1）. It is

ples. After thawing, a diamond cut saw（MC-130D,

considered that raising people’s awareness of oral dis-Maruto Instrument Co., Ltd., Tokyo, Japan）was used toease prevention through esthetics may improve their

cut the cervical area and incisal portion under water

quality of life. However, teeth bleaching is not perma-

injection. The central enamel part of the cervical area

nent, and the teeth color may gradually return to thewas excised. After cutting, the enamel surface on the2）state prior to treatment . Pigmentation on the toothlabial side of the crown was polished with water-resis-

surface due to the consumption of colored food andtant abrasive paper（#1,000）such that the polished2）

drinks may cause color reversal of whitened teeth .

surface was parallel to the measurement table. This

Therefore, it is recommended to refrain from consum-

was used as the standard polished surface for the anal-

ing colored foods and drinks to prolong the bleachingysis of enamel surface roughness.

effect2）.

2 ．Preparation of immersion solution

Nowadays, many foods and drinks come in various

Turmeric（TM）（S&B turmeric［powder］, S&B Foods

colors. Studies on color preference for foods haveInc., Tokyo, Japan）, red pepper（RP）（S&B red pepper

revealed that people tend to gravitate towards warm

［powder］, S&B Foods Inc.）, and tomato（TO）（La

colors, such as yellow and orange3,4）. Therefore, color

Preziosa［peeled tomatoes in tomato juice］, Camel Cof-

directly affects interest and motivation in consuming

fee Co., Ltd., Tokyo, Japan）, which are ingredients of

3,4）. These studies emphasized the highcurry and reportedly affect teeth, were used toimportance of food and drink color, and so seasonings,

immerse the enamel samples. Milli-Q water（MQ）was

spices, coloring agents, etc. are used to add color.

used as a control. A 2％ aqueous solution dissolved in

Depending on the condition of the oral cavity, consum-

MQ was used for turmeric and red pepper. Each sam-ing them in large amounts can cause a change in color

ple was immersed in 30 mL of the solution7）. The sam-of the teeth.

ples in each group were randomly assigned to color

foods and drinks

Factors that affect a change in tooth color can bedivided into extrinsic and intrinsic factors 1,5）. Thechange analysis（n＝5）or enamel surface roughness
analysis（n＝7）.

effects of drinks on tooth staining have already been

3 ．pH measurement

discussed1,5,6）. Exogenous coloring can be caused by pig-

The pH of the immersion solution was evaluated byments in fluids, such as coffee, wine, black or green tea,measuring the solution before and after immersion

and tobacco tar6）. In addition, curry is a well-known

using a pH measuring instrument（Desktop pH meter

cause of exogenous coloring. However, the mechanism

LAQUA F-71, Horiba, Ltd., Kyoto, Japan）. Measure-by which foods cause tooth staining has not been eluci-

ments were performed after calibrating using standard

dated. Dental care professionals must instruct patientssolutions. The temperature of the immersion solution

on how to prevent exogenous staining and color rever-during measurement was kept at 37℃ using a ther-sion after teeth bleaching and list the foods that are

mometer, which was the same temperature as whenlikely to cause staining.

the sample was colored. Three measurements wereThus, in this study, we used bovine teeth to determine the effects of colored foods on the enamel surface.
The null hypothesis was that tooth staining of theenamel surface is not affected by dietary changes.

obtained for each immersion solution, and the averagevalue was calculated as the pH value.
4 ．Staining method
One enamel sample was immersed in 30 mL of each
immersion solution in an incubator at 37℃ for 3 hours
while being shaken. After immersion, the samples weretaken out, immediately washed thoroughly with dis-ODEP Vol. 3, No. 1

82

tilled water, dried naturally, and then measured forTable 1color changes and surface roughness.
5 ．Obser vation method
1 ）Color changes
Colorimetry was performed using a micro-area spectrophotometer（MSP-300H, Nippon Denshoku Industries
Co., Ltd., Tokyo, Japan）before and after immersion in
the immersion solution. Colorimetry was performed atfive points in the center of each sample, and the aver-Results of the pH measurements（mean±SE）
before treatment

after treatment

6.903±0.077
6.603±0.001
5.616±0.004
4.446±0.000

6.965±0.031
6.920±0.027
6.009±0.008
4.464±0.012

MQ
TM
RP
TO

MQ：Milli-Q water, TM：turmeric, RP：red pepper, TO：
tomatoage value was used as the measured value. Using the
CIE1976L＊a＊b＊ color system, the measurement results＊

Results＊

were obtained for L , which represents lightness, a ,which represents the saturation of reds and greens, and
＊

1 ．pH

b , which represents the saturation of yellows andTable 1 shows the pH before and after immersion.

blues. Furthermore, the differences between the values

MQ and TM showed near neutral pH, while RP and TO

before and after immersion were defined as ΔL＊, Δa＊and Δb＊, and the standard error was calculated. More-over, ΔE ab, which expresses color difference, was＊obtained using the following formula:

ΔE ab＝［（ΔL ）＋（Δa ）＋（Δb ）］
＊

＊ 2

＊ 2

＊ 2 1/2

The change in color was measured.
2 ）Enamel surface roughness by 3D laser micro-

both showed acidic pH. Particularly, the pH of TO waslowest among the four, well below the critical pH. No
significant change in pH was observed before and after
immersion in each solution at 37℃.
2 ．Color changes
Figures 1 to 3 show the results of comparison
between groups by considering the difference between
the values before and after immersion.

scope
Surface roughness was measured using a 3D laser

There was no significant difference in ΔL＊ between

microscope（LEXT OLS4000, Olympus Co., Tokyo,

the MQ group and all other groups（Figure 1）. A signif-

Japan）. Arithmetic mean roughness（Sa）denotes theicant difference in Δa＊ was observed between the MQ

surface roughness, and the reference polished surface

group and all other groups（Figure 2）. There was a

was measured at five points per sample. The averagesignificant difference in Δb＊ between the MQ and TM

value±standard error was calculated. The measure-groups, and between the MQ and RP groups. However,ment area was set to 645×645μm, and the cutoff value

there was no significant difference between the MQ

7）

was set to 80μm . Sa0 denotes the reference value
before treatment, and Sa1 the value after treatment.

and TO groups（Figure 3）.
Figure 4 shows the measurement results of the color

6 ．Statistical processing

difference value（ΔE＊ab）. ΔE＊ab, which expresses theRegarding the color change, normality was confirmed

color difference（i.e., change in color）, was highest in the

between the four groups of color difference values by

TM group. No significant difference was observed

Dunnett’s multiple comparison test
（p＜0.05 or p＜0.01）.

between the MQ group and all other groups.

In cases where normality could not be confirmed, Steel’s
multiple comparison test was performed.

3 ．Enamel surface roughness by 3D laser microscope

In the test of surface roughness value, the paired

Figure 5 shows the surface roughness measure-t‒test was performed for those with a normal distribu-

ments. Changes in Sa values before and after immer-

tion. The Wilcoxon signed-rank sum test was per-sion in each group were different in all groups（p＜formed when normality could not be confirmed（p＜0.05）. Moreover, the Sa values after immersion signifi-

0.05）
.

cantly increased in the TO group.

Discussion
Nowadays, many foods and drinks come in variousDec, 2023

Δ L＊
10
9
8
7
6
5
4
3
2
1
0

The Effects of Dietary pH Changes on Tooth Staining of the Enamel Surface

ΔE ＊ab
12
10
8
6
4
2
0
MQ

Fig. 1

TM

RP

TO

MQ

Fig. 4

Results of the color changes for ΔL＊
values

Surface roughness（Sa μm）

＊＊

RP

TO

Results of the color difference value for
ΔE＊ab

＊

0.25

＊

0.20

＊

0.15

＊
＊

0.10

MQ

Fig. 2TMRP＊：p＜0.05TO＊＊：p＜0.01

Results of the color changes for Δa

Δb＊
6

0.05
0

MQ

＊

valuesFig. 5

TM
Sa0

Sa1

RP

＊

TO
：p＜0.05

Results of the surface roughness measurements

Sa0：before treatment, Sa1：after treatment.

＊＊
＊＊

5

conjectured that the pH of each food is related to the4

pH of drink, and that the intake of a large amount of3

colored foods may have some effect on the color

2changes of teeth and enamel surface. Consequently, in

1
0

TM

0.30

＊
＊

Δa
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

83

this study, we investigated the effects of colored foods
MQ

TM

RP

on the enamel surface by dietary pH changes.

TO＊＊：p＜0.01

Fig. 3Results of the color changes for Δb＊values

1 ．Literature on foods that cause tooth staining
Curry and vegetables reportedly cause tooth staining 11,12）. Ghai et al. 11） examined the possibility thatIndian food contains ingredients that cause tooth erosion（acid erosion）, and thereby cause tooth staining.

colors. In particular, coffee, wine, and tea are likely to

They reported that ingredients in Indian cuisine havecause teeth staining8）. Tea products, which are con-erosive effects, and that some ingredients, such as

sumed by all ages from children to adults, include caf-tomatoes and red pepper powder, may erode enamel.

feine, polyphenols, and tea stains, which are well-known

Moreover, some ingredients showed decreased pH withcauses of coloration9）. Furthermore, frequent intake ofincreased cooking temperature. These findings suggest

10）

water with low pH demineralizes teeth . On the otherthat these spices used in cooking affect tooth staining,

hand, some foods use a lot of coloring. Therefore, we

and that pH also affects the surface roughness of

ODEP Vol. 3, No. 1

84

enamel.

of the enamel surface. In this study, the TO group had

2 ．Color change based on food characteristics（pigment component）

The Δb＊ value showed a tendency to increase in all

a rougher enamel surface than the MQ, TM, and RP
groups. This was due to the fact that the pH of the TO
group was 4.0, which was well below the enamel criti-

groups, and a significant difference was observed

cal pH of 5.5, suggesting that demineralization of the

between the MQ group and these groups, suggesting

enamel surface increased the roughness.

an increase in the yellow color. Leu et al. reported that

In addition, a significant difference was observed in

the yellow component of turmeric in curry is curcumin,

the MQ group before and after immersion. This may

which is a diketone natural pigment13）. In this study, thehave been due to the surface roughness of the sample

curcumin in turmeric acted to increase the Δb＊ value,

used in the experiment; further verification is requiredin the future.

which indicates yellow color.
Red peppers, or hot peppers, contain a carotenoid
that gives them a red color called capsanthin. However,

4 ．Interaction between tooth surface and pigmentcomponents

capsanthin is also contained in paprika and is a natural

In addition to pigments, interaction between thepigment that gives an orange-to-red color14）. This sug-

tooth surface and pigment component causes color

gests that the reason why the Δb＊ values changed inchange. Nathoo19） reported that the coloring in foods,

the RP group was that these values were also affecteddrinks, and tobacco is associated with ionic bonds,

by the action of capsanthin, a natural pigment, whichhydrogen bonds, and hydrophobic interactions, and col-changed the color to orange.

oration occurs when the calcium in the oral cavity and

The TO group is similar to the RP group. Tomatoes

saliva-derived proteins are oxidized and calcified. In thiscontain a natural carotenoid pigment called lycopene,

study, although the immersion time was as short as 3

which changes the color from orange to red

15）. Thismay be the same reason for the change in Δb value in＊hours, it was speculated that calcium, which is a component of teeth, bonded with curcumin and capsanthin,

the RP group, since this value may have been affectedcausing a color change. Thus, changes in teeth color are

by lycopene, based on the color change to orange. In

caused by the pH of the foods and drinks that are nor-addition to tomatoes, beets are considered to bemally consumed in daily life, demineralization of teeth

12）

reported

due to pH, outflow of calcium that occurs due to thethat people with discolored teeth consume large quanti-demineralization, action of saliva in the oral cavity, andties of beets. Moreover, beets contain a betacyani-so forth. Therefore, it is necessary to consider the fre-din-based natural pigment called betanin16）. In thisquency of intake of turmeric and red pepper and otherstudy, we focused on turmeric, red pepper, and tomato

oral conditions when addressing changes in tooth color.

and observed the color change. Spices and vegetables

In addition, whitened teeth are more susceptible tocontain various pigments, and also have various charac-

staining than normal teeth since the surface layer of theteristics, such as oil solubility, color change depending

enamel becomes rougher after bleaching6,20）. However,on pH, and resistance to heat and acid. This suggests

this study did not consider the surface roughness ofthat the characteristics of natural pigments contained

teeth after bleaching; it is necessary to investigate thein food must be considered when assessing color

roughness of the enamel surface after bleaching andchange.

how this affects the change in teeth color.

involved in tooth staining. de Araújo et al.

3 ．C o l o r c h a n g e b a s e d o n t h e r e l a t i o n s h i p

Curry and vegetables reportedly cause tooth staining.
Curry is often cooked using turmeric, red pepper,

between tooth surface and pH
Regarding enamel surface roughness, there was a

tomato, and various other spices. Mixing various foods

difference before and after immersion in the MQ, TM,

may cause color changes and enamel surface rough-and RP groups. In particular, in the TO group, the val-

ness. During immersion, color changes and enamel sur-ues tended to increase after immersion, suggesting thatface roughness may occur depending on changes in thethe enamel surface had become rough. The critical pH

pH of food due to temperature and immersion time. In

at which enamel demineralizes is 5.5
et al.

10）

17,18）. Tahmassebi

this study, the temperature was set to 37℃, andreported that low pH drinks may cause erosion

immersion was performed for 3 hours. Therefore, it isDec, 2023

The Effects of Dietary pH Changes on Tooth Staining of the Enamel Surface

necessary to investigate the change in pH with temperature rise and immersion time in the future.
In this study, we conducted an experiment to clarify
the effects of spices and vegetables on teeth; however,these have several positive effects on the body. For
example, turmeric is well-known to improve liver function. It is important to understand the characteristics ofeach food and provide instructions to patients regarding
their esthetic and health demands, rather than refraining from eating and drinking because of tooth staining.

Conclusion
This study was conducted to clarify the effects offood on the enamel surface of teeth. The results sug-gest that the Δa＊ value, which represent redness, andthe Δb＊ value, which represents yellowness, increased

due to the influence of the natural components in turmeric and red pepper. Tomato had a pH lower than theenamel critical pH of 5.5, suggesting that it may be
involved in the roughening of the enamel surface.
Conflicts of Interest
The authors declare no conflict of interest related to thispaper.
Acknowledgements
This work was supported in part by the FUTOKU foundation（2022）for T.E.

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MEJ. Soft drinks and dental health: a review of the current literature. J Dent 2006; 34: 2‒11.
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Fraga Briso AL, Fraga Zaze ACS, Sundfeld RH. Mineralloss and color change of enamel after bleaching andstaining solutions combination. J Biomed Opt 2013; 18（10）: 108004.
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antitumorigenic effect of curcumin. Curr Med Chem
Anticancer Agents 2002; 2: 357‒370.
14）Kennedy LE, Abraham A, Kulkarni G, Shettigar N, DaveT, Kulkarni M. Capsanthin, a plant-derived xanthophyll:a review of pharmacology and delivery strategies.
AAPS PharmSciTech 2021; 22: 203.
15）Story EN, Kopec RE, Schwartz SJ, Harris GK. An updateon the health effects of tomato lycopene. Annu Rev
Food Sci Technol 2010; 1: 189‒210.
16）Gliszczyńska-Swigło A, Szymusiak H, Malinowska P.
Betanin, the main pigment of red beet: molecular originof its exceptionally high free radical-scavenging activity.
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18）Stephan RM. Intra-oral hydrogen-ion concentrations
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19）Nathoo SA. The chemistry and mechanisms of extrinsic
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ODEP Vol. 3, No. 1

86

Original Article

ODEP 3（1）：86～94，2023

Evaluation of the Hardness of Infected Root Canal Dentinwith Fluorescence Excitation
Shuya SHIBANO, Mie MAYERS, Tokuji HASEGAWA1, Takashi TAKAKI2 and Katsuhiko ISATSU
Department of Perioperative Medicine, Division of Medical and Dental Cooperative Dentistry,
Showa University School of Dentistry1

Department of Conservative Dentistry, Division of Comprehensive Dentistry, Showa University School of Dentistry2

Center of Electron Microscopy, Showa University

Abstract
Purpose: This study aimed to evaluate the association between infection and hardness in root canal dentin
by irradiating infected root canal dentin with blue light at a wavelength of approximately 405 nm.
Methods: A total of 10 healthy human extracted teeth and 10 human extracted teeth with infected dentin
were used as samples and semi-cut sections were prepared as longitudinal sections of the root canal. The
root canal walls were observed using a stereo microscope under blue light at a wavelength of approximately
405 nm to measure the fluorescence spectrum and Vickers hardness. Subsequently, the area was observed
using a scanning electron microscope.
Results: In teeth with infected root canals, blue-light irradiation at a wavelength of approximately 405 nm
induced fluorescence excitation at wavelengths of approximately 620 and 680 nm. In the healthy teeth, nosignificant difference was observed in Vickers hardness in the region with fluorescence excitation of up to
500μm from the root canal wall; however, in the infected teeth, a significant decrease in Vickers hardness
was observed in the region with fluorescence excitation of up to 500μm from the root canal wall. Furthermore, in the infected teeth, a bacterium-like structure was observed via electron microscopy on the root
canal wall surface, and in the dentin tubules in areas with fluorescence excitation, whereas in areas without
fluorescence excitation, there were few bacterium-like structures.
Conclusion: This study suggests that blue light of approximately 405-nm wavelength is useful in detecting
infected areas in the root canal wall.
Key words: blue light, infected root canal dentin, Vickers hardness

Corresponding author: Katsuhiko ISATSU, Department of Perioperative Medicine, Division of Medical and Dental Cooperative
Dentistry, Showa University School of Dentistry, 2‒1‒1, Kitasenzoku, Ota-ku, Tokyo 145‒8515, Japan
TEL: ＋81‒3‒3787‒1151（ext. 248）, FAX: ＋81‒3‒3787‒1229, E-mail: isatsu@dent.showa-u.ac.jp
Received for Publication: September 15, 2023/Accepted for Publication: November 6, 2023
DOI: 10.11471/odep.2023‒010

Dec, 2023

Relationship between Hardness and Fluorescence in Root Canal Dentin

87

As an extension of these studies, the present study
aimed to evaluate the usefulness of blue light in the

Introduction

treatment of infected root canals by visualizing infected
Apical periodontitis is a lesion in which bacterial

areas of root canal dentin using blue light and showing

infection in the root canal causes periodontal tissue

bacterial erosion of infected areas using Vickers hard-

inflammation and destruction. It is generally known

ness testing and electron microscopy.

that many bacteria inhabit the oral cavity. In healthy
teeth, on the other hand, the pulp is sealed within the

Materials and Methods

dentin, and thus cannot be infected by bacteria. Basically, the pulp of a healthy tooth cannot be infected, but

1 ．Specimen preparation

if for some reason a pathway between the pulp and theIn this study, human extracted teeth were used fororal cavity is established, infection can occur. Pathway

sample preparation. The study protocol was approved

development is thought to be mainly caused by either

by the ethics committee of Showa University（21-004-

pulp exposure due to caries, fissures, or trauma, or by

A）. Tooth extractions were performed at Showa Uni-

dentin tubules in the dentin exposed by enamelversity Dental Hospital, or Showa University Fujigaoka

1,2）

destruction. Pulp infection caused by a pathway for

Hospital.

oral bacteria results in pulpitis, and as the infection pro-Due to the limited number of healthy teeth available

gresses, the pulp vasculature is compromised, resulting

as a control group, no restrictions were placed on thein pulp necrosis. If left untreated, this condition can leadtypes of teeth used in this study. When collecting theto apical periodontitis, in which pulp cavity infection

extracted teeth, we collected teeth with apical peri-

spreads beyond the root apex and destroys the sur-odontitis, in which infection of the root canal was

1）

expected, and vital teeth with periodontitis or pericoro-

However, it is difficult to observe the inside of thenitis of wisdom tooth, in which the tooth structure wasrounding periodontal tissue .

root canal with the naked eye, and infected dentin may

not infected.

remain in the root canal, causing recurrence of apicalThe extracted teeth were immersed in 10％ neutral

periodontitis. Although microscopes enable precise

buffered formalin and stored at room temperature forobservation of the inside of root canals, they are expen-

disinfection so as not to affect hardness8）. Subsequently,

sive, huge, not easily portable, and also require skill andthe teeth were rinsed with sterile purified water, and a

training to operate. Even if a root canal can besemi-cut section was prepared by cutting the root canalobserved using a microscope, there is currently no indi-

longitudinally under water injection using a diamondcator to show which part of the canal wall is infected.

disk. To prepare the specimens, the cut surface wasThus, the current practice is to mechanically enlarge

polished with water-resistant abrasive paper to a

the root canal using hand cutting instruments, or nick-roughness of #2000 under water injection.

el-titanium rotary files, chemically clean the canal withendodontic irrigants such as sodium hypochlorite

3,4）

, and

apply calcium hydroxide preparations to sterilize theroot canal3‒5）.

2 ．Obser vation of fluorescence using a stereo
microscope under blue light
The polished surface was placed on a glass slide witha flat pressure machine so that it was horizontal, andOur previous studies focused on the fact that red flu-

the surrounding area was fixed with self-polymerizing

orescence excitation was observed when infected den-resin. Then, the samples were observed using a stereo

tin was irradiated with blue light of approximately 405-

microscope under blue light of approximately 405-nm

nm wavelength. We observed that bacteria were pres-wavelength in a dark room. A total of 10 teeth wereent in infected areas where red fluorescence excitation

allocated to each group: the infected group of samples

was seen. Furthermore, when red fluorescence exci-

in which red fluorescence was observed in the root

tation was observed by irradiating a paper point with acanal wall, and the healthy group of samples in whichroot canal wiped with blue light of approximately 405-

no red fluorescence was observed.

nm wavelength, the tooth in question was found to be
6,7）

infected in a bacterial culture test.

3 ．Fluorescence measurement
After observation under the stereo microscope, theODEP Vol. 3, No. 1

88

the root canal for each of the 10 teeth was used to
semiconductor laser

determine the extent to which differences in hardness
by distance from the root canal caused by differencesin dentin tubule density in the measurement range,
should be considered in this study.
5 ．Evaluation of Vickers hardness of the root
canal wall in infected root canals

darkroom

spectroscopic analyzer

Microscopic fluorescence measurement system
The sample was irradiated with blue laser light, theinduced fluorescence was analyzed using a spectrometer（MCPD7700, Otsuka Electronics Co., Ltd., Tokyo,
Japan）, and the fluorescence spectrum was recorded.
Fig. 1

Vickers hardness was then determined at the fluorescence spectrum measurement sites and the change
in Vickers hardness before and after the boundary
where fluorescence excitation occurred was evaluated.
Hardness measurement of root canal dentin has beenpreviously used to investigate the effects of endodontic
irrigants and lubricants on the dentin. The Vickers
hardness test, which is commonly used for this purpose,
was also employed in the present study11,12）.
The receiver operating characteristic curve was cre-healthy group was first examined using a microscopic

ated using the hardness of root canal wall dentin influorescence measurement system（Fig. 1）to measure

healthy and infected teeth to determine the extent to

the red fluorescence spectrum produced by blue light ofwhich infection weakens the hardness of the root canalapproximately 405-nm wavelength when irradiated atwall dentin.

distances of 100, 300, 500, and 700μm from the root
canal wall. To confirm the absence of red fluorescence,

6 ．Obser vation of samples using a scanning electron microscope

the fluorescence spectrum was measured at 10 points

Half of the infected teeth were observed using an

along the root canal wall. In the infected group, the flu-

electron microscope（FlexSEM 1000, Hitachi High-Techorescence spectrum at 100μm before and after theCorp., Tokyo, Japan）. Formalin adhering to the sample

boundary between the infected and healthy areas was

was washed by agitation with phosphate-buffered saline

measured at 10 points along the root canal wall.

for 10 min. The samples were then dehydrated with

4 ．Evaluation of Vickers hardness of the root

ethanol at concentrations of 50％, 60％, 70％, 80％, 90％,99％, 100％, and again 100％, in that order, for 10 min

canal wall in the healthy group
Because dentin tubules generally run radially fromeach. Next, critical point drying using a critical point

the pulp side to the enamel-dentin boundary and root

dryer（931.GL, Tousimis, Maryland, USA）was per-surface, the closer they are to the outside, the lower

formed on the samples. The sample was then fixed totheir density per unit area, and the greater their hard-the sample table with carbon tape so that the observa-

ness and the closer they are to the pulp side, the higher

tion surface was as horizontal as possible, and degassed

9,10）. There-in a vacuum until the next day. Finally, the sample sur-fore, to elucidate the hardness decrease of the root

face was coated with osmium using an osmium coater

canal wall due to infection and the hardness change of（HPC-1SW, Shinkuu Device Co., Ibaraki, Japan）, andthe root canal wall due to the difference in dentin

observed using an electron microscope.

their density and the lower their hardness

tubule density, we measured the Vickers hardness of

7 ．Statistical analysis

the fluorescence spectrum measurement sites near the

Vickers hardness of the root canal wall in healthyroot canal wall of healthy teeth using a microhardness

teeth was evaluated by Scheffé’s method. Statisticaltester（FM-300, Future-Tech Corp., Kanagawa, Japan）.

analysis was conducted using EZR software（Saitama

The Vickers hardness test was performed with a load

Medical Center, Jichi Medical University, Saitama,of 300 gf and a loading time of 10 seconds. Further-

13）
Japan）. The differences between the results obtained

more, we determined whether the difference was negli-from the two methods were assessed using Fischer’s

gible. Using healthy teeth, hardness by distance fromexact probability test and analysis of variance. Vickers

Dec, 2023

Relationship between Hardness and Fluorescence in Root Canal Dentin
Table 189

Vickers hardness of the root canal dentin by distance from the canal wallin healthy teeth

Distance from root canal wall

Number

Minimum

Maximum

Mean

SD

100μm
300μm

10
10

38.06
42.57

55.32
53.8

45.6

5.7

500μm
700μm

10
10

45.03
51.41

59.46
74.51

49.3
52.8
63.7

3.74
4.33
6.57

Vickers hardness of the root canal wall was measured using sound teeth todetermine if there was a significant increase or decrease in the range of root
canal wall hardness measurements.
Vickers hardness was measured at distances of 100, 300, 500, and 700μm fromthe root canal wall in healthy teeth, and the mean value of each distance was calculated. As a result, Vickers hardness increased with increasing distance fromthe root canal wall, particularly at a distance of 700μm from the canal wall, andthe increase in hardness was significantly greater than those in other areas.

hardness of the root canal wall with and without fluo-

Fluorescence spectrum measurements showed a spe-

rescent excitation in infected root canals was evaluatedcific fluorescence spectrum with two peaks at approxi-

by the t-test.

mately 620- and 680-nm wavelengths in the vicinity of
the infected root canal wall（Fig. 3）. A peak was

Results

observed at approximately 500 nm, similar to that of thehealthy teeth. Away from the root canal wall, a wave-

1 ．Healthy teeth

form similar to that of the healthy teeth was observed.

Red fluorescence was not observed when the healthy

Fluorescence spectra were recorded at 100μm before

group was irradiated with blue light. When blue light ofand after the boundary where the specific fluorescenceapproximately 405-nm wavelength was irradiated atspectrum disappeared at a distance of 500μm from the

distances of 100, 300, 500, and 700μm from the root

root canal wall. Furthermore, Vickers hardness wascanal wall and the fluorescence spectrum was mea-

measured at the fluorescence spectrum measurement

sured, a waveform with a peak wavelength of approxi-

site, and the average hardness before and after themately 500 nm was observed; however, the specificboundary was calculated. The results indicated that inwaveform seen in infected tooth structure was not

all 10 specimens, the hardness was lower in the area

observed. Vickers hardness was measured at the fluo-

with the specific fluorescence spectrum than in therescence spectrum measurement site and the averagearea without（Table 2）.

value of each sample was calculated according to the

In the infected teeth, Vickers hardness was used to

distance from the root canal wall. The results indicatedevaluate the degree of attenuation in the area with fluo-that Vickers hardness increased with increasing dis-

rescence excitation compared with the area withouttance from the root canal wall except for one of the 10

fluorescence excitation as a percentage. Vickers hard-samples（Table 1）. In that outlier sample, Vickers hard-

ness at distances of 100, 300, and 500μm from the root

ness at 100μm from the root canal wall was slightly

canal wall measured on a healthy tooth was used tohigher than those at 300 and 500μm. The results indi-

calculate the percentage difference in hardness forcated no significant differences between the distances

every 200μm. The proportion of both groups wasof 100‒300, 300‒500, and 100‒500μm, but a significant

included in the receiver operating characteristic curve,

difference was observed between the distance of 700

and the optimal cutoff value that maximized the sum of

μm and all other distances（p＜0.05; Scheffé’s method）.
2 ．Teeth with infected root canals
When the infected group was irradiated with blue
light, red fluorescence excitation was observed（Fig. 2）.

specificity and sensitivity was determined to be 91.9％（Fig. 4）.
3 ．SEM
After measuring the fluorescence spectrum andODEP Vol. 3, No. 1

90fluorescence intensity

0.03

a

0.02

0.01

b

Fig. 2 Findings with incident light and blue lighta）The boundary between the infected and healthydentin could not be distinguished when the samples
were observed under a stereo microscope. b）When
blue light was irradiated, red fluorescence wasobserved from the infected dentin（arrow）, and theboundary between the infected and healthy dentin was
clearly defined.

Infected root canal dentin
Healthy dentin

0

350 400 450 500 550 600 650 700 750 800 850
wavelength（nm）

Fluorescence spectrum of infected and healthydentin in the root canal dentin
The fluorescence intensity on the vertical axis andthe wavelength on the horizontal axis were plotted ona graph of the fluorescence excitation generated byblue-light irradiation of the samples. Specific waveforms were observed at approximately 620 and 680 nm

Fig. 3in the infected root canal dentin. No specific waveforms were observed in the healthy dentin.
Table 2

Vickers hardness before and after the boundary between the areas
with and without fluorescence excitation in healthy teeth

Fluorescence excitation
Presence
Absence

Number

Minimum

Maximum

Mean

SD

10
10

20.22
27.12

52.3
58.33

37.6
43.4

9.77
10.02

To examine whether the presence or absence of infection causes significant
hardness attenuation in the root canal wall, using a tooth with an infectedroot canal, Vickers hardness was measured at a distance of 100μm before
and after the boundary where a specific fluorescence spectrum disappeared
at a distance of 500μm from the root canal wall, and the respective averagevalues before and after the boundary were calculated. The hardness of thearea with a specific fluorescence spectrum was significantly lower than thatof the area without（p＜0.05）.

Vickers hardness of the infected group, half of themwere observed under an electron microscope. Bacteri-

Discussion

um-like structures were seen on the surface of the root
canal wall and in the dentin tubules of the indentations

In recent years, based on the philosophy of minimalin areas with fluorescence excitation. On the otherintervention dentistry proposed by the FDI World Den-hand, few bacterium-like structures were observed intal Federation, it has been recommended that dental

the dentin tubules of the indentations in areas withoutcaries treatment should focus on the protection offluorescence excitation（Fig. 5）.

healthy tooth structure14）. In the treatment of infectedroot canals, the protection of healthy dentin can reduce
the risk of root fracture and help extend the life of theDec, 2023

Relationship between Hardness and Fluorescence in Root Canal Dentin

91

specific fluorescence spectrum with two peaks at

1.0

approximately 620 and 680 nm was observed. No specific fluorescence spectrum was observed in the root

0.919（0.600, 0.800）

0.8canal walls of the healthy teeth, or at sites away from

Sensitivity

the root canals of the infected teeth. These results suggest that blue light of approximately 405-nm wave-

0.6

length is useful for detecting infected root canal dentin
and dentin caries in the crown.

0.4

The hardness of the root canal wall of healthy teeth
was measured and the results were evaluated. The

0.2

results showed that only the area 700μm from the root
canal wall had significantly higher hardness than theother measured areas（p＜0.05）. Therefore, it was sug-

0.0

1.0

0.8

0.6
0.4
Specificity

0.2

0.0

Fig. 4 ROC curve based on the rate of change ofVickers hardness
A receiver operating characteristic（ROC）curve
was created by combining the rate of hardness
change for every 200μm of the healthy tooth andthe rate of hardness change between 200μm
across the boundary between the infected andhealthy areas of the infected root canal tooth. The
cutoff value was derived from the ROC curve tomaximize the sum of sensitivity and specificity.

gested that a hardness comparison between 100 and
500μm is worth considering, although the hardnessesof 100, 300, and 500μm are not exactly equal in healthy
teeth.
Based on these results, fluorescence spectra at 100
μm before and after the boundary where the specificfluorescence spectrum disappears up to 500μm fromthe root canal wall were recorded at 10 points eachalong the root canal wall using an infected tooth, andVickers hardness at that site was measured. For eachsample, the average of Vickers hardness before andafter the boundary was calculated and a t-test was conducted. The hardness of the area with a specific fluo-

tooth. For this purpose, it is effective to visualize therescence spectrum was significantly lower than that of

infected root canal wall using some methods and selec-

the area without（p＜0.05）. Therefore, in the areas

tively remove the infected area instead of blindly grind-

where fluorescence was observed, the dentin was con-ing and mechanically expanding the root canal wall assidered to be weakened by the root canal infection.

in the current method for treating infected root canals.

The optimal cutoff value for hardness reduction due

In this study, the presence or absence of bacterial

to root canal infection obtained from the receiver oper-

infection in root canal dentin, which emits red fluores-

ating characteristic curve generated was 91.9％. This

cence excitation when irradiated by blue light ofsuggests that if there is a boundary between the pres-approximately 405-nm wavelength, was examined via

ence and absence of fluorescence within a distance offluorescence spectrum and Vickers hardness measure-

500μm from the canal wall in an infected tooth, thement as well as microstructural observation using anhardness is approximately reduced to 91.9％ or lesselectron microscope. Our previous studies have demon-

before and after the boundary. Thus, if the percentage

strated that bacterial infection occurs in crown dentinof hardness change is above 91.9％ after removal of 200

with red fluorescence excitation in response to blue

μm of the root canal wall, it can be inferred that thelight; however, the effect of blue light on root canal den-healthy dentin has been approximately reached.

tin has not been investigated. The present study aimed

Electron microscopy of infected root canals showedto evaluate the effectiveness of blue light on infected

the presence of bacterium-like structures on the root

root canals in relation to hardness.

canal wall surface and in the dentin tubules at theIn the infected root canal dentin and crown dentin

indentations in areas with fluorescence excitation, butcaries, red fluorescence excitation was produced by

few bacterium-like structures were observed in areas

blue light of approximately 405-nm wavelength, and awithout fluorescence excitation, suggesting that the

ODEP Vol. 3, No. 1

92

a

b

c

d

Fig. 5 Scanning electron microscope（SEM）image of a tooth with infected root canal
The root canal wall surface layer of an infected tooth and the site where the Vickers hardness
test was conducted were observed using an electron microscope.
a ）Root canal wall surface：the presence of bacterium-like structures was observed（arrow）.
b ）Dentin tubules in the area with specific fluorescence spectrum：the presence of bacterium-likestructures was observed（arrow）.
c ）Dentin tubules in the area without specific fluorescence spectrum：bacterium-like structures
were rarely observed.
d ）Stereo microscopic image of the areas observed with SEM.

presence of bacteria contributes to the occurrence of

Future studies should also determine whether

fluorescence excitation and weakening of the root canalremoval of root canal dentin with fluorescence exci-

dentin. However, there appeared to be a difference intation has a superior therapeutic effect.

size between the bacterium-like structures present on

In this study, Vickers hardness was used as an indi-

the root canal wall surface and those present in thecator of weakening of root canal dentin caused by bac-dentin tubules. Since the bacteria present may differ

teria, but nanoindentation is an indicator of microstruc-depending on the location of the root canal wall dentin,tural weakening due to caries15）. Future studies should

future studies should use PCR techniques to identify

consider using nanoindentation.

the bacterial species present.

This experiment was conducted extraorally using

The Vickers indenter may sometimes fail to measureextracted teeth; it would be difficult to apply this test

the hardness of the unlined area near the surface of themethod directly to actual clinical practice. Therefore,root canal wall due to cracking or sinking of the sur-

instruments to observe the inside of root canals androunding area. Thus, it is necessary to improve the

new instruments for cutting the root canal wall shouldmeasurement direction, for example, by seeking the lin-be developed and the clinical application of blue light to

ing of the tooth structure.

root canals should be considered.

Dec, 2023

Relationship between Hardness and Fluorescence in Root Canal Dentin

93

infection in apical periodontitis and its relationship with

Conclusion
In this study, we measured the fluorescence of
infected dentin in the root canal wall using a microscopic fluorescence measurement system, measured theVickers hardness at the site, and observed the microstructure of the indentation via electron microscopy.
The results suggest that blue light produces red fluorescence excitation in root canal wall infected dentin as
well as dentin caries. This study demonstrated thatblue light at a 405-nm wavelength may be useful for
detecting infected sites in root canals. In actual clinicalpractice, it is difficult to observe the inside of a root
canal using techniques such as those in this study.
Therefore, incorporation of blue light into a dental
endoscope that can be inserted into the root canal,
which we studied, can be applied to clinical practice16）.
However, for curved root canals, it is difficult to use a
dental endoscope or selectively remove the infectedareas even if they can be observed using a dental endoscope. Therefore, further study of the hardness relationship between infected and healthy dentin as well as
the development of an instrument that can blindly andselectively polish hardness below the threshold
between infected and healthy dentin in the root canalwall will facilitate the establishment of more treatments
for infected root canals based on minimal intervention
dentistry. Thus, future research should focus on twoareas: identification of the infected area in the root
canal wall via fluorescence excitation, and removal of
the infected dentin using the hardness threshold as a
guide.
Acknowledgments
We would like to thank the dentists of our department for
their cooperation in collecting the teeth and the staff of theCenter of Electron Microscopy for their help in observing
the specimens.
Conflicts of Interest
The authors declare no conflict of interest related to thispaper.

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14）FDI World Dental Federation. FDI policy statement on

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（in Japanese）

Dec, 2023

95

Original Article

ODEP 3（1）：95～103，2023

Effect of an MDP-based Cleaner on Adhesion to Dentin Contaminatedwith a Temporary Luting Cement
Taiho IDONO, Shojiro SHIMIZU, Tomohiro TAKAGAKI,
Michael F. BURROW＊ and Toru NIKAIDO
Department of Operative Dentistry, Division of Oral Functional Science and Rehabilitation, Asahi University School of Dentistry＊

Restorative Dental Sciences, Faculty of Dentistry, University of Hong Kong

Abstract
Purpose: The aim of this study was to investigate the effect of temporary luting cement removal using a
new 10-methacryloyloxydecyl dihydrogen phosphate（MDP）-based cleaner（KATANA Cleaner）on the dentin adhesion of a resin cement.
Methods: Intact human dentin was ground with 600-grit SiC paper and divided into three groups. The firstgroup was left untreated with the temporary cement（Con）. For the other groups, dentin surfaces were covered with a temporary luting cement（Temporary Hard）and stored in water for one week. The temporary
luting cement was removed using an ultrasonic scaler（US）, and then treated with KATANA Cleaner for
10 （s KC）. A resin composite block（KATANA Avencia Block）was bonded to the dentin using a resin cement（Panavia V5）. The specimens were immersed in water for 24 h then cut into beams. These were subjectedto thermal cycling stress for 0 cycles（TC0）or 5,000 cycles（TC5000）. A microtensile bond strength test was
performed at a crosshead speed of 1 mm/min. Data were statistically analyzed with two-way ANOVA andt-test with Bonferroni correction at a significance of 5％. The fractured specimens after debonding were
examined using scanning electron microscopy and electron diffraction spectroscopy.
Results: No significant differences were noted in microtensile bond strengths between Con-TC0 and
KC-TC0, between KC-TC0 and KC-TC5000 and between Con-TC5000 and US-TC0. The temporary luting
cement covered the dentin surface in the US group, and partially remained in the KC group. Energy-dispersive X-ray spectrometric analysis revealed a distinct Zn peak in the US group, while the peak was significantly decreased in the KC group.
Conclusion: The combined method with an ultrasonic scaler and the MDP-based cleaner effectively
removed temporary luting cement contaminants on dentin surfaces, thus securing durable bonding of a resin
cement to dentin used for indirect restorations.
Key words: MDP-based cleaner, dentin bond strength, resin cement

Corresponding author: Shojiro SHIMIZU, Department of Operative Dentistry, Division of Oral Functional Science and Rehabilitation, Asahi University School of Dentistry, 1851, Hozumi, Mizuho, Gifu 501‒0296, Japan
TEL & FAX: ＋81‒58‒329‒1442, E-mail: shojiro0914@dent.asahi-u.ac.jp
Received for Publication: September 1, 2023/Accepted for Publication: November 9, 2023
DOI: 10.11471/odep.2023‒011

ODEP Vol. 3, No. 1

96

10-methacryloyloxydecyl dihydrogen phosphate（MDP）-containing cleaner, was developed to remove

Introduction

contaminants such as saliva, blood and temporary luting
Recently, increasing demand among patients for

cements/filling materials. This study investigated theesthetic restorative treatments has boosted the needeffect of the MDP-based cleaner on the bonding of afor metal-free crown restorations1）. Computer-aided

resin cement to dentin contaminated with a temporary

design/computer-aided manufacturing（CAD/CAM）-

luting cement.

based restorations have become more popular 2,3）,
mainly with ceramic and resin-based composite materi-

Materials and Methods

als4,5）. Since CAD/CAM resin composites and ceramics
are more brittle than metal alloys6,7）, the clinical suc-

1 ．Materials used in this study

cess of these restorations is greatly enhanced by inte-

The materials used in this study are listed in Table 1.

gration of the restorative materials with the underlying

A resin cement, Panavia V5（Kuraray Noritake Dental,

tooth structure. Otherwise, there is a risk of fracture orTokyo, Japan）was used with the associated self-etchingdetachment of the restorations8）.

primer（Tooth Primer, Kuraray Noritake Dental）

In clinical practice, there are several factors that

according to the manufacturer’s instructions. A phos-

may inhibit adhesion during clinical procedures, espe-

phoric acid etchant（K-Etchant Syringe, Kuraray Nori-cially for indirect restorations9,10）. Saliva contamination

take Dental）and a silane coupling agent（Clearfil

has been reported to be a major factor reducing theCeramic Primer Plus, Kuraray Noritake Dental）were

long-term durability of the adhesion of resin cement toused for the pretreatment of a CAD/CAM resin block

the fitting surface of CAD/CAM resin blocks11）. A tem-

（KATANA Avencia Block, Kuraray Noritake Dental）.

porary crown is generally placed with a temporary lut-

A polycarboxylate-based temporary luting cement

ing cement until the next patient visit to protect the（HY-Bond Temporary Cement, Hard, Shofu, Kyoto,prepared dentin and pulp from bacterial infection, sup-Japan）was used, which is commonly used by Japanese

12）

press hypersensitivity, and prevent tooth movement .

clinicians. A MDP-containing cleaner（KATANA

However, the temporary cement is not easily removed

Cleaner, Kuraray Noritake Dental）was used for remov-

before the final cementation, contaminating the adher-

ing the contaminants from the temporary luting cement

end and reducing the adhesion of the resin cement toon the dentin surface.

12,13）

dentin. Therefore, contamination by the temporary

luting cement must be prevented to ensure clinical success of metal-free restorations.

2 ．Microtensile bond strength test
Specimen preparation procedures for the microtensile bond strength（μTBS）test are illustrated in Fig. 1.

A previous study investigated the mechanicalThis study was approved by the ethics committee ofremoval of temporary cement with an ultrasonic scaler,

Asahi University School of Dentistry（No. 25148）. Intact

but no significant effect was noted compared to thehuman molars were cut perpendicular to the long axis

14）
control（no mechanical cleaning）. In contrast, anotherof the teeth to expose the superficial dentin, which waspaper reported that cleaning off a temporary cement

ground with 600-grit SiC paper to standardize thewith a rotating cone-shaped brush under water-spraysmear layer. Vinyl tape（100μm thick）was placed on

effectively removed the contaminants, recovering thethe dentin surface to ensure a uniform thickness of theadhesion to dentin14）. Such variable results indicate that

cement space.

the overall outcome might also depend on the type of

The specimens were divided into the following threeresin cement used. Cleaning the temporary cement

groups. The dentin surface was left without temporary

with a cone-shaped brush alone was insufficient15）. As

cement in the control group（Con）. For the experimen-

no practical method for removing a temporary cement

tal groups, a polymethyl methacrylate（PMMA）block16）

has yet been established , there is an urgent need to

was placed on the dentin surface with a temporary

establish clinical procedures for effectively removing

cement（HY-Bond Temporary Cement, Hard）and

temporary luting cements to achieve reliable adhesion.

stored at room temperature for 30 min for complete

Recently, a phosphate-based functional monomer,

setting of the cement. The specimens were then storedDec, 2023

Effect of MDP-based Cleaner on Bonding Durability to Contaminated Dentin
Materials used in this study

Table 1
Material

Manufacturer

KATANA Avencia Block

Kuraray Noritake

97

Composition

Lot. No

Alumina filler（20 nm）, Silica filler（40 nm）, Resin monomer

769

MDP, HEMA, Hydrophilic aliphatic dimethacrylate, Accelerators, Water
Paste A：Bis-GMA, TEGDMA, Hydrophobic aromatic dimethacrylate, Hydrophilic aliphatic dimethacrylate, Initiators,
Accelerators, Silanated barium glass filler, Silanated fluoroalminosilicate glass filler, Colloidal silica
Paste B：Bis-GMA, Hydrophobic aromatic dimethacrylate,Hydrophilic aliphatic dimethacrylate, Silanated barium glassfiller, Silanated alminium oxide filler, Accelerators, dl-Camphorquinone, Pigments
3-Methacryloxypropyl trimethoxysilane, MDP, Ethanol

34007

Dental, Tokyo, Japan
Panavia V5

Clearfil Ceramic Primer
Plus
KATANA Cleaner
HY-Bond Temporary
Cement（Hard）

K-Etchant Sylinge

Shofu, Kyoto, Japan

Kuraray Noritake
Dental, Tokyo, Japan

390130390130

1P0051

Water, MDP, Triethanolamine, Polyethyleneglycol, Accelerator, Dyes

CGO004

Powder：Zinc oxide, Silica, Magnesium oxide, Tanninfluoride
compound, Colorant etc.
Liquid：Acrylic acid-tricarboxylic acid copolymer, Sodium salt,
Purified water

91916

Phosporic acid（35％）, Polyethyleneglycol, Colloidal silica,
Water, Pigment

42016
570141

Bis-GMA；Bisphenol A di（2-hydroxy propoxy）dimethacrylate, HEMA；2-Hydroxyethyl methacrylate, MDP；Methacryloyloxydecyl, dihydrogen phosphate, TEGDMA：Triethylene glycol methacrylate.
Untreated (Con)40s light-cured(VALO, 1,000 mW/cm2)
Double-sided
tape

PMMA
block

#600 SiC

Human tooth

Failure mode
analysis under SEM
observation
Dentin surface witha temporary cement

Ultrasonic scaler(US)

Bonding withresin cement

Stored in
distilled water at
37℃ for 24h

Sectioned into
1 mm-squarebeam shapeμTBS at 1 mm/min
cross-head speed
EZ-SX (Shimadzu)
n=20

Thermal cycle 5,000
between 5℃ and 55℃(TC 5000)KATANA Cleaner(KC)

Fig. 1

Specimen preparations for μTBS testing and SEM/EDS analysis

in water at 37℃ for one week. Then, the PMMA blocks KC）.
tionally scrubbed with KATANA Cleaner for 10 （was manually removed with a dental explorer. An

All specimens were rinsed with distilled water andultrasonic scaler（ENAC OE-3, Osada, Tokyo, Japan）

dried just before the bonding procedure.

was used in the output mode 2 for 10 s to remove any

The CAD/CAM resin block was cut into sections ofvisible contaminants on the dentin surface using careful

2-mm thickness with a low-speed cutting machine

visual examination（US）. The dentin surface was addi-（Isomet1000, Buehler, Lake Bluff, IL, USA）and groundODEP Vol. 3, No. 1

98with #600-grit SiC paper under a stream of water. The
surface of the resin block was abraded with 50-μm alumina（DANVILLE, San Ramon, CA, USA）at a distance

Resin
block

of 10 mm with an air pressure of 0.2 MPa immediately
before the bonding procedure17,18）. Subsequently, theresin blocks were sonicated in 99.9％ ethanol for 2 minARand air-dried. Next, phosphoric acid etch（K-EtchantSyringe）was applied on the surface for 5 s, followed by

CementAD＋CR

rinsing with distilled water and air-drying. A silane cou-ADpling agent（Clearfil Ceramic Primer Plus）was finallyapplied to the specimen surface for 10 s and gently air-

AD＋CD

CD

dried.
For the dentin side, a self-etching primer（Tooth

DentinPrimer）was applied to the dentin surface for 20 s andair-dried. A resin cement（Panavia V5）was then applied
to the dentin surface and the CAD/CAM block wasplaced and pressed onto the dentin surface. A LED
light curing unit（VALO Curing Light, ULTRADENTPRODUCTS, South Jordan, UT, USA）was used in thestandard mode（1,000 mW/cm2）on the top of the CAD/
CAM block to directly cure the resin cement for 40 s
through the CAD/CAM block. Afterwards, the bondedCR

Illustration of categorization of the fracturemodes after debonding
AD：Adhesive failure at the interface between thedentin and resin cement
CR：Cohesive failure in the resin cement
AR：Adhesive failure at the interface between theresin cement and CAD/CAM resin block
CD：Cohesive failure in the dentin
Fig. 2

specimens were kept at room temperature for 30 min,and stored in water at 37℃ for 24 h. The specimens

4 ．SEM and EDS analysis of the dentin surface

were cut perpendicular to the dentin-resin cement

The dentin surface of each group was prepared ininterface to make beam-shaped specimens（1 mm×1

the same manner as the specimens prepared for themm）using a low-speed diamond cutting machineμTBS test. The dentin surfaces were osmium-coated,（Isomet 1000）. The specimens were then subjected toand observed using an SEM. In addition, elemental anal-

thermal stress at 5‒55℃ for 30 s dwell time using ayses of Ca, P, and Zn were performed using an ener-

thermal cycling machine（K178, Tokyo Giken, Tokyo,

gy-dispersive X-ray spectrometer（EDS, EMAX-7000,

Japan）for 0 cycles（TC0）or 5,000 cycles（TC5000）,

Horiba, Tokyo, Japan）attached to the SEM at an accel-

respectively. A μTBS test was performed using a uni-

eration voltage of 1.5 kV.

versal testing machine（EZ-SX, Shimadzu, Kyoto, Japan）

5 ．Statistical analysis

at a crosshead speed of 1 mm/min.

The number of specimens was 20 for each group.

3 ．Fracture mode analysis

The data obtained from the μTBS test were analyzed

The fracture modes of the debonded specimens afteru s i n g t h e S h a p i r o―W i l k t e s t f o r n o r m a l i t y a n dμTBS testing were examined using a scanning electron

Levene’s test for homogeneity of variance. Statistical

microscope（SEM, S-4500, Hitachi, Ibaraki, Japan）after

analysis was then performed using two-way analysis of

the fractured specimens were osmium-coated（HP-IS,

variance（ANOVA）and the Bonferroni-corrected t-test

Vacuum Devices, Horiba, Mito, Japan）. The fracture

at a significance level of 5％.

modes were categorized as follows: adhesive failure at
the interface between dentin and resin cement（AD）,
cohesive failure in the resin cement（CR）, adhesive fail-Results

ure at the interface between the resin cement and1 ．Microtensile bond strength

CAD/CAM resin block（AR）, cohesive failure in dentin

Table 2 shows the results of the μTBS test. Two-（CD）and mixed failure including the combinations ofway ANOVA analysis revealed the interaction between

AD＋CR and AD＋CD as illustrated in Fig. 2.

“material”and“thermal cycling.”There were no sig-Dec, 2023
Table 2

Effect of MDP-based Cleaner on Bonding Durability to Contaminated Dentin
Microtensile bond strengths of dentin
with/without thermal cycles（TC）

Group
ControlUSKC

TC0
51.2±12.8AB

35.0±9.851.8±10.1A

99

the control group and a peak for Zn was clearly identified. The Zn peak was also identified in the KC group,

TC5000

but the amount of Zn in KC was much smaller thanBthat in US, while the amounts of P and Ca in KC wereC

relatively higher than those in US.

36.1±8.5

24.7±7.852.6±14.5A

N＝20, Mean（MPa）±SD
Same superscript letters indicate no significant
difference（p＞0.05）. Thermal stress for 0（TC0）and 5,000 cycles（TC5000）.

Figure 7 shows the elemental mapping images foreach group. In the control group, P and Ca belonging todentin apatite were detected on the entire surface. In
the US group, P and Ca were also detected, and Zn wasstrongly detected, matching the area of the temporary
cement residues on the dentin surface observed in thenificant differences in μTBS between Con-TC0 and

SEM images. Small amounts of Zn were also detected

KC-TC0, between KC-TC0 and KC-TC5000, andin various sites of the SEM images in the KC group.

between Con-TC5000 and US-TC0, respectively.
2 ．Obser vation of fractured surface morphology

Discussion

Figure 3 shows representative SEM images of thefractured surfaces after the μTBS test. Figure 4 shows

In this study, we investigated the effect of removing

the different fracture mode percentages for the speci-

temporary cement contaminants from the dentin sur-mens for each group. The fracture mode was mainly

face using an ultrasonic scaler and MDP-based cleaner.

cohesive failure in the resin cement（CR）in all groups,

We also evaluated the dentin bonding performance

except for KC-TC5000. Compared to Con-TC0, adhesive

when a CAD/CAM resin block was bonded to dentin

failure（AD）was more commonly observed in US-TC0

with a resin cement. The temporary cement, HY-bondand KC-TC0. Mixed failure including cohesive fracture

Temporary Cement（Hard）, is a polycarboxylate-based

in dentin（AD＋CD）was observed only in the control

cement which is widely used for temporization for indi-groups. For the KC groups, both adhesive failure

rect restorations. The output modes of the ultrasonic

between the resin cement and dentin（AD）and mixed

scaler range from mode 1 to mode 4, which is a varia-

failure（AD＋CR）tended to increase from TC0 totion in intensity, mode 4 being the strongest. The opti-

TC5000.

mal output mode was determined in a pilot study. The

3 ．SEM obser vation of dentin-bonded surface

results were as follows: mode 1- the sonication was too

Figure 5 shows the secondary electron images of the

weak to remove the temporary material sufficiently,

dentin surface of each group. For the control group, theand residual cement was visually confirmed; mode 2-

smear layer and the scratch marks created by the 600-

contaminants from the temporary cement were entirely

grit SiC paper were observed on the dentin surface.

removed by visual inspection; modes 3 and 4- sonication

For the US group, residual temporary cement covered

was too strong and was found to damage the dentin

the entire dentin surface. For the KC group, scratch

surface, with scratches from the scaler tip chipping themarks were observed the same as the control group,

dentin surface. Therefore, output mode 2 was selected

however, small amounts of temporary cement residue

for this experiment.

were noted. In addition, dentinal tubule openings were

The MDP-containing cleaner, KATANA Cleaner, was

not observed with the partial removal of the smear

tested in this study. MDP is a functional monomer usedlayer.

in many adhesive systems. MDP is known to play a

4 ．EDS analysis of the dentin surface for eachgrouprole in self-etching dentin in the presence of water,
resulting in stable chemical bonding with the calcium in

Figure 6 shows the SEM images（Upper）
and the EDS

hydroxyapatite of human teeth19‒21）. On the other hand,line analysis（Lower）of the Con, US, and KC groups,

MDP has a hydrophilic group and a hydrophobic grouprespectively. From the EDS analysis, the two peaks ofin one molecule, and so has the possibility to exert a

P and Ca belonging to dentin were confirmed in all

surfactant effect. In this study, the action of MDP in KC

groups, which decreased in the US group compared towas believed to be due to the surfactant effect. The

ODEP Vol. 3, No. 1

100

Fig. 3 Representative SEM images of the fractured specimens after the μTBS testing
CR：Cohesive failure in the resin cement
AD：Adhesive failure at the interface between the dentin and resin cement
AD＋CD：Mixed failure of AD and cohesive failure in the dentin（CD）（×70）.
The white arrows in the SEM images indicate the boundary between AD and CD.

temporary cement used in this study has hydrophobic

Con－TC0characteristics. When KC is applied on the contami-

US－TC0nated dentin surface, MDP probably forms a micellestructure by surrounding and dispersing the hydropho-

KC－TC0bic temporary cement. The micelle structures can be

Con－TC5000

easily removed and washed out.

US－TC5000

The control group without contamination revealed
the highest bond strength of 50 MPa at TC0, however,

KC－TC5000
0%
AD

20%
40%
60%
AR
CR
AD+CR

80%
100%
AD+CD

Results of the failure mode analysis after
debonding of specimens
AD：Adhesive failure at the interface between thedentin and resin cement
CR：Cohesive failure in the resin cement
AR：Adhesive failure at the interface between theresin cement and CAD/CAM resin block
CD：Cohesive failure in the dentin
Fig. 4the bond strength decreased in US, whereas the bondstrength was recovered in KC to the same value asthat of the control group. This strongly suggested thatremoving the temporary cement with the combinationof ultrasonic scaler and the MDP-based cleaner would
be very effective before bonding of a resin cement todentin.
After the thermal cycling, the bond strengths
decreased in the Con and US groups. In contrast, nodecrease was found in the KC group, suggestingDec, 2023

Effect of MDP-based Cleaner on Bonding Durability to Contaminated Dentin
US

Control

101

KC╳1,000╳2,500╳5,000Fig. 5

Representative SEM photographs of the dentin surface after removal of the temporary filling material

Control

US

KC

Zn

Ca
P
Ca

P

Zn

O

O
Ca
Fig. 6

P

Ca

Ca

O
Ca Zn

Zn

SEM（Upper）and EDS line analysis（Lower）of the dentin surface for each group

improvement of dentin bond durability with the KC

ure in resin cement（CR）in all the groups, except fortreatment, which may be due to the surfactant effect

KC-TC5000. The rate of adhesive failure at the resin

and also the formation of MDP-Ca salt by application of

cement-dentin interface（AD）increases at TC5000. In

KC. The results of the fracture mode analysis（Fig. 4）

KC-TC5000, AD and AD＋CR were mainly observed,

showed that the main fracture mode was cohesive fail-which is believed to be due to the effect of the

ODEP Vol. 3, No. 1

102
SEM

O

P

Ca

Zn

Control

US

KC

Fig. 7

EDS mapping analysis of the dentin surface for each group

KATANA Cleaner on the dentin surface, but the main
reason remains unclear. Further morphological investi-

Conclusion

gations of the bonded interface should be performed.
The SEM image of the dentin surface in the control

The combined method with an ultrasonic scaler andgroup（Fig. 5）showed that the dentin surface was

the MDP-based cleaner effectively removed contami-

entirely covered with a smear layer, while the residues

nants from a temporary luting cement on dentin sur-of the temporary cement remained in the US group,

faces, thus securing durable bonding of a resin cement

despite complete removal of the contaminants con-to dentin used for indirect restorations.

firmed by visual inspection. From the EDS analysis（Figs. 6 and 7）, Zn was detected, which must have

Conflict of Interest

been derived from zinc oxide（ZnO）in the temporary

The authors declare no conflicts of interest associatedwith this manuscript.

cement in the US group. The remarkable reduction of
the Zn peak in the KC group（Fig. 6）suggested that theresidual temporary cement on the dentin surface of KC
is much less than that of US, however, KC could notcompletely remove all of the contamination caused by
the temporary cement. Therefore, it is concluded thatthe combination of an ultrasonic scaler and KC could
remove the temporary cement efficiently, resulting in
improvement of bonding between the resin cement anddentin.
Only one type of temporary cement material wasused in this study. Further studies should be undertaken to investigate the effect of different types of temporary cements to establish a reliable method forremoving contaminants from temporary luting cements.

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K. Temporary cement remnants as an adhesion inhibit-

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ODEP Vol. 3, No. 1

104

Original Article

ODEP 3（1）：104～112，2023

Color Evaluation of Porcelain Laminate Veneer Restorations
Bonded with a Single-shade Resin Composite
Toshinari TOYAMA, Mikihiro KOBAYASHI, Rintaro SUGAI, Yuiko NIIZUMA and Atsufumi MANABE
Department of Conservative Dentistry, Division of Aesthetic Dentistry and Clinical Cariology,
Showa University School of Dentistry

Abstract
Purpose: To evaluate the effect of single-shade resin composites on the color of porcelain laminate veneer（PLV）restorations comprising lithium disilicate（LDS）cemented to an underlying tooth of two different
shades. The evaluation was carried out using the CIEDE2000 color-difference formula and a translucency
parameter.
Methods: Ceramic blocks（A2, HT, IPS e.max CAD, Ivoclar）of 1 mm thickness were fired and assumed to
be PLV. Their adhered surfaces were polished with grit-silicon carbide paper（#1200）, and their measurement surfaces were mirror-polished. Four resin composites were used as luting materials to fill the 50-μm
space between the PLV and the 2-mm-thick standardized underlying tooth, which was then photo-polymerized to serve as the specimen. The following luting materials were used in the present study: single-shade
GRACEFIL Universal LoFlo（GC）, Clearfil Majesty ES Flow（Kuraray Noritake Dental）, Omnichroma Flow
（Tokuyama Dental）, and A2 shade GRACEFIL LoFlo（GC）. A2 and A4 resin composites（Filtek Supreme
Ultra, 3M ESPE）polished with water-resistant polishing paper（#600）were assumed as the underlying tooth（underlying structure）. Forty specimens were produced and immersed in distilled water at 37℃ for 30 days.
There were two conditions for the color of the underlying structure and four conditions for the luting material（n＝5）. A Vitae Easy Shade V（VITA）digital spectrophotometer was used to determine the color ofeach specimen. The PLVs were investigated before and after cementation, and after immersion in water（Day 1, 3, 7, and 30）on the underlying structure. The data were analyzed using the 1976 CIEL＊a＊b＊ color. ΔE00 was calculated for the underlying structure spacing and compared to asystem and CIEDE2000（ΔE00）. The translucency parameter（TP）, deterthreshold considered acceptable in clinical practice（ΔE00＞1.8）
mined by the color difference between the black and white backgrounds, was also evaluated. The statisticalsignificance of each of the ΔE00 and TP values at Day 30 was determined by the Shapiro-Wilk and Levene
tests and one-way analysis of variance（ANOVA）.
Results: The one-way ANOVA results showed no significant differences in the ΔE00（p＝0.32）or TP（p＝0.23）values between the luting materials at Day 30. However, the ΔE value between the underlying structure of A2 and A4 exceeded the threshold value（ΔE00＞1.8）.
Conclusion: The present study suggests that the color of the underlying tooth has more influence on the
final color of the PLV restoration than the single-shade resin composite.
Key words: porcelain laminate veneer, single-shade resin composite, color

Corresponding author: Mikihiro KOBAYASHI, Department of Conservative Dentistry, Division of Aesthetic Dentistry and Clinical Cariology, Showa University School of Dentistry, 2‒1‒1, Kitasenzoku, Ota-ku, Tokyo 145‒8515, Japan
TEL: ＋81‒3‒3787‒1151, FAX: ＋81‒3‒3787‒4146, E-mail: mkobayashi@dent.showa-u.ac.jp
Received for Publication: September 15, 2023/Accepted for Publication: November 9, 2023
DOI: 10.11471/odep.2023‒012

Dec, 2023

Color of Laminate Veneer Restorations Bonded with a Single-shade Resin Composite

105

ites that use pigments to express color, the filler content, shape, and size have a photochemical effect andIntroduction

blend with the color of the surrounding tooth strucPorcelain laminate veneer（PLV）restorations are one

ture13）. Therefore, the present authors hypothesizedof the most commonly used esthetic treatments forthat using a single-shade resin composite as a luting

tooth discoloration and morphological abnormalities, andmaterial in PLV restorations would suppress the influ-

are relatively minimally invasive. In addition, PLV isence of luting material on the final PLV color, rendering

clinically highly popular among patients because of its

the procedure simple and esthetic.

superior esthetics, optical properties, and biocompatibil-

In this study, PLVs were placed on the underlying

ity1‒8）. Studies examining 21- to 25-year survival rates of

tooth by using a single-shade light-cured resin compos-

feldspathic porcelain laminate veneers reported highite, and color changes were measured with a spectro-

1）survival rates and long-term stability . In recent years,

photometer. Spectrophotometers are often used to eval-lithium disilicate ceramics（LDS）have been widely useduate the color of PLVs after restoration in vitro

in PLV restorations because of their excellent esthetic

because they can standardize and quantify color; theproperties2）. The color of the final PLV restoration

color difference（ΔE00）by using CIEDE2000 indicates amight be influenced by the thickness and translucencyof the veneer restoration, depending on the amount ofchange in color, and clinically, ΔE00 of ＞1.8 indicates adominant color discrepancy14）. In several studies, alight scattering and reflection 8）. It has also beenpost-cementation ΔE00 of ＞1.8 was considered a clini-

reported that the color of the resin cement has a signif-cally unacceptable color discrepancy. It is also import-

icant influence on the color of the restoration after

ant to provide natural tooth-like transparency in treat-

cementation because of the high translucency of thement with LDS15）. Appropriate transparency enables

LDS. Furthermore, it has been reported that the color

PLVs to appear natural16）. The translucency parameterof the underlying tooth also greatly affects the overall（TP）is frequently used to measure the translucency ofcolor of the PLV restoration3）. Because such resto-

ceramics17）. Therefore, in this study, the influence of therations are generally fabricated with a PLV thickness

luting material on the translucency of PLVs was alsoof 0.5‒1.0 mm4）, the color of the resin cement inter-evaluated by using the TP. Although there have been

posed between the translucent LDS and the underlying

studies on post-cementation coloration of PLV resto-

tooth can have an important influence on the clinicalrations using LDS, there are few studies on the effect of5）

success of the restoration .

single-shade resin composites as a bonding material

Dual-cure resin cements are widely used for cemen9）

between the underlying tooth and PLV on the color-tation of ceramic restorations . However, theseation of PLV restorations. The purpose of this in vitro

cements have a short working time and can cause color

study was to evaluate the effect of resin composites

changes over time, especially yellowing of the cementwith a wide range of color compatibility on the color of

because of oxidation of reactive groups of tertiary

PLV restorations by using the color difference andamines and polymerization inhibitors, which mighttransparency.

affect the color after PLV restoration

10,11）. Therefore,

light-cured resin composites are sometimes used toMaterials and Methods

12）bond PLV to enamel . Such composites are available
in a variety of shades and restore human teeth, which

Table 1 shows the materials used in this study. IPS

express many shades of color. However, clinician skill

e.max CAD
（IvoclarVivadent, Schaan, Liechtenstein）, an

and experience are necessary to select the appropriate

LDS-based ceramic block, was used as the PLV. The

shade for each individual tooth and achieve an esthetic

shade of the ceramic block was selected to be A2, a

restoration. Therefore, single-shade resin composites

highly translucent type. The underlying teeth were pre-with a simple shade system have been developed andpared by using Filtek Supreme Ultra（3M ESPE, St.

clinically used in resin composites in recent years. The

Paul, MN, USA）in A2 or A4, and the PLV was bonded

characteristic feature of color reproduction with mono-

to the underlying tooth by using a single-shade flowable

chromatic resin composites is that unlike resin compos-

resin composite. GRACEFIL Universal LoFlo（GUF: GC,

ODEP Vol. 3, No. 1

106
Table 1

Materials used in this study
Main composition

Manufac-

Code
Resin composite（Luting material）
GRACEFIL LoFlo
CNT

Filler con-

Lot Number

turer

Matrix

Fillertents（wt％）

GC

Bis-MEPP

Barium glass

69

2212151

GRACEFIL Univeral
LoFlo

GUF

GC

Bis-MEPP, TEGDMA,
UDMA

Silicon dioxide, Strontium glass

692304281

Clearfil Majesty ES
Flow

ESF

Kuraray
Noritake
DentalTEGDMA

Barium glass filler,
Silanated silica, Nanocluster filler

75CC0052

Omnichroma Flow

OCF

Tokuyama
DentalUDMA, TEGDMASiO2-ZrO2 filler

71

042B3

Bis-GMA, UDMA,
Bis-EMA, TEGDMAS i O2 - Z r O2 n o n - a g -78.5

A2：9906824
A4：9849212

Resin composite（Underlying structure）
Filtek Supreme Ultra

3M ESPE

glomerated filler

Ceramic block（PLV）
IPS e-max CAD

IvoclarVivadent

Lithium disilicate
glass-ceramic（SiO 2 ,Li2O, K2O, P2O5, ZrO2,

Z00VL9

Al2O3, MgO）

Tokyo, Japan）, Clearfil Majesty ES Flow（ESF: Kurarayture surfaces were polished with water-resistant abra-

Noritake Dental, Tokyo, Japan）, Omnichroma Flow

sive paper（#600）. After polishing, the PLVs and under-（OCF: Tokuyama Dental, Tokyo, Japan）, and GRACE-

lying tooth were ultrasonically cleaned for 10 min and

FIL LoFlo（CNT: GC）
in the A2 shade were used. These

dried. As a pretreatment for adhesion, the surfaces offour types of light-curing flowable resin composites

the underlying structure and the PLV were sand-were used as luting materials.

blasted for 5 s using sand with an average grain size of

1 ．Specimen preparation

50μm at a pressure of 0.3 MPa. They were then coatedTo prepare the specimens, IPS e.max CAD was pre-with a silane coupling agent（Clearfil Ceramic Primer

pared to a thickness of 1 mm by using an automaticPlus, Kuraray Noritake Dental）. Aluminum spacers（50precision cutting machine（Isomet, Buehler, Lake Bluff,

μm thick）
were fixed between the PLVs and underlying

IL, USA）and fired in a fast sintering furnace（CEREC

structure, and luting material was loaded and photo-po-

SpeedFire, Dentsply Sirona, Bensheim, Germany）.

lymerized. Light irradiation was performed on one side

After firing, the surface polished with grit-silicon car-of the specimen in two sections for 30 s each, for a total

bide paper（#1200）was assumed to be the adherendof 120 s from both sides, and the polymerized speci-

and the surface was mirror-polished with aluminummens were used as the test specimens. A total of 40

oxide powder（0.05μm: MICROPOLISH II, Buehler was

specimens were prepared and then immersed in dis-assumed as the color measurement surface. The toothtilled water at 37℃ for 30 d.

was fabricated with A2 and A4 resin composites,

2 ．Color analysis

adjusted to a 2-mm-thick rectangle using a silicone

Specimens were colorimetrically measured with aguide, and photo-polymerized and assumed as thedigital spectrophotometer（Easy Shade V, VITA Zahn-

underlying tooth（underlying structure）. Light irradia-

fabrik, Bad Säckingen, Germany）before bonding thetion was performed by using the normal mode of a light

PLVs to the underlying structure［before cementationi r r a d i a t o r（G - L i g h t P r i m a I I P l u s , G C）w i t h a n（BC）］; immediately after cementation with luting mate-8.0-mm-diameter irradiation port at a light intensity ofrial（Day 0）, and at 1, 3, 7, and 30 days after immersion2

1,200 mW/cm for 90 s each side. The underlying struc-in water. Colorimetry was performed on a black back-Dec, 2023

Color of Laminate Veneer Restorations Bonded with a Single-shade Resin Composite

ground under room light at the same time and location.
The specimens were guided to define the color measurement site and angle to enable reproducible color
measurement. Before each color measurement, the107over black and white backgrounds, respectively.
4 ．Statistical analysis
ΔE00 and TP values for each specimen at Day 0‒30

were calculated as mean±standard deviation（SD）. The

specimens were calibrated as specified by the manufac-

normality and homoscedasticity of the ΔE00 and TP val-

turer; the same area was measured three times and theues at Day 30 were verified using the Shapiro-Wilk andaverage value was used as the measured value. The

Levene tests, and analyzed by one-way analysis of vari-color was analyzed by using the 1976 CIE L＊a＊b＊

ance（ANOVA）only between luting materials in each＊color system to obtain lightness L （white to black）,
a＊（red to green）, and b＊（yellow to blue）. The overall

specimen. ΔE00 between A2 and A4 underlying structure and ΔE00 because of cementation were comparedcolor was calculated by using CIEDE2000（ΔE00）. The

with an acceptable threshold（ΔE00＞1.8）. The data

difference in color between the underlying structure of

were analyzed at a significance level of 0.05 using theA2 and A4 was also calculated by using ΔE00 . Each

JMP Pro 17 software package（SAS Institute, Cary, NC,

ΔE 00 was compared with the threshold consideredUSA）.

acceptable in clinical practice（ΔE00＞1.8）. The parameters and formulas for calculating ΔE00 are shown in a

Results

subsequent paragraph. The difference（ΔL′）, saturationdifference（ΔC′）, and hue difference（ΔH′）were calcu-

1 ．Color analysis

lated to compute ΔE0018,19）（0 is the value immediately

The Shapiro-Wilk and Levene tests were used toafter cementation, and n is the value measured on each

assess the normality of ΔE00 at Day 30 in each speci-

measurement day）.

men, indicating that the data were normally distributed

ΔL′ ＝Ln－L0

and homoscedastic. One-way ANOVA indicated no sig-

ΔC′ ＝［（an）＋（bn）］ －
［
（a0）＋
（b0）］
2

2 1/2

2

2 1/2

2
2
2 1/2
ΔH′ ＝［（Δa）
＋
（Δb）
－
（ΔC）
］

［（KΔL′S ）＋（KΔC′S ）＋（KΔH′S ）
2

ΔE00＝

2

L L

C C

（KΔC′S ）（KΔH′S ）］ ,

＋RT

2

H H

1/2

C C

H H

nificant difference in ΔE00 between the different luting

materials（p＝0.32）. Figures 1 and 2 show changes overtime in specimens for each condition, Table 2 showseach parameter from 0‒30 days, and Table 3 shows
ΔE00. Figures 3 and 4 show graphs of E00 for 0‒30 days.

The ΔE00 values for each condition at Day 30 were:

where SL , SC , and SH denote the heavy value coeffi-

4.5±0.5 for CNT, 4.7±0.6 for GUF, 4.8±0.2 for ESF,

cients；the Patrick factors for KL, KC, and KH were set

and 4.7±0.6 for OMC for the A2 underlying structure;

to 1.

and 4.3±1.1 for CNT, 4.5±0.8 for GUF, 4.5±0.3 for

The colorimetric conditions were two conditions for

ESF, and 4.3±0.6 for OMC for the A4 underlying struc-different colors of the underlying structure and fourture. The ΔE00 values before and after cementation

conditions for different luting materials（n＝5）.

were: 4.7±0.5 for CNT, 5.7±1.2 for GUF, 4.5±0.8 for

3 ．Transparency analysis

ESF, and 4.5±0.4 for OMC for the A2 underlying struc-

Translucency is a visual assessment of a material’sture; and 6.4±1.9 for CNT, 6.9±1.4 for GUF, 6.2±1.3

masking ability, or the extent to which the material canfor ESF, and 6.8±1.2 for OMC for the A4 underlying

reveal the appearance of the underlying background.

structure. All of these values were greater than the

The TP is determined by calculating the color differ-acceptable threshold（ΔE00＞1.8）. Table 4 shows theence between a black and white background for speci-

ΔE00 values of the color difference between the under-mens of the same thickness.

lying structure of A2 and A4: CNT was 5.5±0.5, GUF

In this study, low foam vinyl chloride boards（Acrysunday, Tokyo, Japan）
with black and white back-was 4.4±0.8, ESF was 5.3±1.2, and OCF was 5.2±0.5
at Day 30; which are all ＞1.8 in all conditions.

grounds were used. The following formula was used for

2 ．Transparency analysis

calculating TP17）.

The Shapiro-Wilk and Levene tests were used to

2
2
2 1/2
TP＝
［
（L＊B－L＊W）＋（a＊B－a＊W）＋（b＊B－b＊W）］ ,evaluate the normality of TP from Day 30 in each spec-

where subscripts B and W refer to color coordinates

imen, indicating that the data were normally distrib-ODEP Vol. 3, No. 1

108
BC

Day 0

Day 1

Day 3

Day 7

Day 30

CNT

GUF

ESF

OCF
Representative image obtained before cementation（BC）, and 30-day
color change of the specimen with the A2 underlying tooth
CNT：GRACEFIL Low Flow, GUF：GRACEFIL Universal Low Flow,
ESF：Clearfil Majesty ES Flow, OCF：Omnichroma Flow
Fig. 1

BC

Day 0

Day 1

Day 3

Day 7

Day 30

CNT

GUF

ESF

OCF
Representative image obtained before cementation（BC）, and 30-day
color change of the specimen with the A4 underlying tooth
CNT：GRACEFIL Low Flow, GUF：GRACEFIL Universal Low Flow,
ESF：Clearfil Majesty ES Flow, OCF：Omnichroma Flow
Fig. 2uted and homoscedastic. A one-way ANOVA indicatedno significant difference in the TP between the luting

Discussion

material for any of the days（p＝0.23）. Table 5 shows
the TP for each condition. The TP values at Day 30 for

Clinically, PLV restorations are affected by the color

each condition were 8.7±2.9 for CNT, 12.4±2.8 forof the resin cement and that of the underlying

GUF, 11.0±0.8 for ESF, and 10.4±1.0 for OCF for the

tooth20,21）. Therefore, it is important to investigate theA2 underlying structure; and 10.6±0.8 for CNT, 9.5±

influence of the color of the resin cement and underly-

2.5 for GUF, 8.1±0.6 for ESF, and 10.4±2.6 for GUF foring tooth used in the final PLV restoration.

the A4 underlying structure.

The CIELAB color space system quantifies color by

Dec, 2023

Color of Laminate Veneer Restorations Bonded with a Single-shade Resin Composite

109

Results evaluated as mean of color parameters（L＊, a＊, b＊）before cementation（BC）and

Table 2over 30 days（n＝5）
L＊

A2

L＊

－1.8
－0.8

11.3
13.4

82.5
75.7

14.3

77.1

14.5
13.2

74.1
73.8

－1.4
－1.1

19.8

74.7

2.6

＊

＊

Day 3

76.0
74.9

－0.7
－1.0

75.7

0.7

＊

BC
Day 0

Table 3

b＊

81.9
75.8
77.9

Day 1
Day 3

－0.9

CNT
＊

L

a

77.5

－1.1

69.5
71.2

0.6
0.8

14.6
14.9
21.0

67.7

GUF

a＊

BC
Day 0
Day 1
Day 7
Day 30

A4

CNT

b

－0.3

Day 7

67.8

－0.3

Day 30

68.3

2.5

ESF

a＊

b＊

L＊

－1.8
－0.7

11.7
14.1

82.4
76.4

15.2

78.5

13.7
13.2

74.8
74.0

－1.8
－1.0

20.4

74.7

1.6

＊

＊

－0.8

GUF
＊

L

a

b

11.5

78.5

－1.3

15.2
16.7

69.8
71.4

0.4
0.6

68.2

－0.4

14.5

68.1

0.1

14.1

68.9

2.1

22.1

OCF

a＊

b＊

L＊

－1.8
－0.9

11.4
13.0

82.2
76.6

14.5

78.7

12.3
13.3

75.2
74.9

－1.9
－1.2

20.0

75.0

1.3

＊

＊

－0.9

ESF
＊

a＊

b＊

－1.8
－1.2

11.8
12.4

－1.2

13.7
11.8
12.5
19.6

OCF

L

a＊

11.7

78.1

－1.1

11.7

14.3
16.3

69.7
71.4

0.3
0.3

68.8

－0.1

15.0
15.7

68.4

0.0

14.2

69.5

2.6

21.5

L

a

b

10.7

78.1

－1.2

15.1
16.2

70.2
71.1

0.2
0.6

68.7

－0.1

14.8

68.0

0.0

13.9

69.2

2.4

22.2

b＊

14.6

Results of color change evaluated as mean ΔE00±SD of specimens at Day 30 of immersion inwater and before and after cementation（n＝5）
Color parameters（ΔE00）
CNT
A2

Day 304.5±0.5
Cementation（Day 0） 4.7±0.5

GUF
A4

A2

ESF
A4

A2

OCF
A4

A2

A4

4.3±1.1

4.7±0.6

4.5±0.8

4.8±0.2

4.5±0.3

4.7±0.6

4.3±0.6

6.4±1.95.7±1.2

6.9±1.4

4.5±0.8

6.2±1.3

4.5±0.4

6.8±1.2

One-way ANOVA indicates no significant（p＝0.32）difference in ΔE00 between the various luting mate-

6

6

5

5

4

4
ΔE00

ΔE00

rials（Line）.

3

2
1.8
1

2
1.8
1
0

CNT

■Day 1

GUF■Day 3

ESF■Day 7

OCF

0

CNT

■Day 1■Day 30

Mean and standard deviation values for ΔE00 at
Day 1, 3, 7, and 30 in the A2 underlying toothColor evaluation after submersion in water for 30days with various luting materials（CNT, GUF, ESF,
OCF）
（n＝5）
. The red dotted line indicates the acceptability threshold（ΔE00＞1.8）.

Fig. 3

3

Fig. 4

GUF■Day 3

ESF■Day 7

OCF■Day 30

Mean and standard deviation values for ΔE00 at
Day 1, 3, 7, and 30 in the A4 underlying toothColor evaluation after submersion in water for 30days with various luting materials（CNT, GUF, ESF,
OCF）
（n＝5）
. The red dotted line indicates the acceptability threshold（ΔE00＞1.8）.

ODEP Vol. 3, No. 1

110using mathematical formulas to objectively determine

were single-shade, with the exception of CNT. OCF

color variation18）. Recently, CIEDE2000（ΔE00）―whichuses structural color to match the color of the tooth23）.

uses saturation and hue to correct the color range per-The structural color of OCF is based on the results ofceived by humans―has been recommended for color

photochemical processes of diffraction, interference, and19）

scattering23,24）. Therefore, they are not supposed to fade,analysis .

The amount of change in each color was analyzed

in contrast to resin composites that use pigments asusing ΔE 00 . However, ΔE 00 can only calculate thecolor24）. However, ESF and GUF―unlike OCF―matchamount of color change and cannot analyze up to thethe color with the surrounding dentin by adjusting lightcolor direction. Therefore, parameters L, a, and b

diffusion and light transmission25,26）. However, in the（which indicate the direction of each color change）

present study, no significant difference in ΔE00 was

were analyzed. This study compared the effect of resin

found between the luting materials, including CNT incomposites（termed single-shade, which can reproduce

the A2 shade. Differences in commercially available lut-

many shades of color）on the color of PLV restorations

ing materials do not affect the color of PLV resto-

when cemented to an underlying structure of two dif-rations27）. In this study, a single-shade resin composite

ferent shades. In this study, ANOVA indicated thatwas used as the luting material, and the results were

there was no statistically significant difference in ΔE00

similar. In this study, the color decreased after PLV

from Day 30 by resin composite type, p＝0.32. Therecementation and a ΔE00 well above 1.8 was observed; inwas also no significant difference in the TP values

PLV restorations, the color difference decreases before

between cements for any of the days and underlying

and after cementation as the ceramic thickness

structure conditions. In areas where the PLV thickness

increases21）. In this study, all PLV specimen thicknesses

is thin, the transparency is high and the saturation

were adjusted to 1.0 mm to standardize the conditions.

might decrease when a luting material with low satura-

Clinically, PLV thicknesses range from 0.5‒1.0 mm, and22）tion is used . The resin composites used in this study

although the results of this study might differ for theminimum thickness, the color change because of cementation was consistent with previous reports3）. The

Table 4 Results of color change assessed by mean
ΔE 00±SD between underlying teeth ofeffect of the difference in the color of the underlying

different color over 30 days（n＝5）
CNTstructure, A2 and A4, on the overall color was investigated; the ΔE00 value between the underlying structure

Color parameters（ΔE00）
GUF

ESF

OCF

exceeded the acceptable threshold in all conditions andwas visually recognizable. This suggests that the differ-

Day 0

5.3±0.8

4.9±0.8

5.0±1.4

5.7±0.8

Day 1

5.5±1.0

5.2±0.7

5.6±1.7

5.6±0.7

ence in the color of the underlying structure, A2 andDay 3

6.3±1.3
4.5±0.5

4.6±1.1
4.7±1.1

5.8±1.7
4.8±0.2

5.8±0.7
4.7±0.6

A4, affected the color of the PLV restorations. As the

5.5±0.5

4.4±0.85.3±1.2

5.2±0.5

masking ability decreases and the color of the underly-

Day 7
Day 30

Table 5

ceramic thickness decreases in PLV restorations, theResults evaluated by mean TP±SD over 30 days（n＝5）
Translucency parameter（TP）

CNT

GUF

ESF

OCF

A2

A4

A2

A4

A2

A4

A2

A4

Day 0

6.2±1.0

6.0±1.96.8±2.0

6.4±2.9

7.0±0.3

5.4±2.6

6.2±2.0

5.2±1.9

Day 1

6.0±0.7

6.5±1.77.5±1.3

7.1±3.1

5.9±0.75.4±2.1

5.9±0.55.4±1.3

Day 3

6.8±2.1

8.4±2.77.8±2.4

8.7±2.88.8±1.0

6.3±3.4

8.2±2.7

6.5±1.4

Day 7

8.6±0.6

7.0±0.8

10.0±1.7

7.1±1.2

9.0±1.2

6.5±3.2

9.4±0.88.6±1.2

Day 308.7±2.910.6±0.8

12.4±2.8

9.5±2.5

11.0±0.8

8.1±0.6

10.4±1.0

10.4±2.6

One-way ANOVA indicates no significant（p＝0.23）difference in TP between the various luting
materials（Line）.

Dec, 2023

Color of Laminate Veneer Restorations Bonded with a Single-shade Resin Composite

111

ing structure as well as the color of the cement areevident28）. In the present study, the color of the under-

Conclusion

lying structure affected the final color when the PLV
was as thick as 1 mm.

In PLV restorations with LDS, differences in the sin-

No significant differences were found between luting

gle-shade resin composite used for cementation did notmaterials in terms of the TP values; a higher TP value

affect the final color and transparency. In contrast, the29）

corresponded to a lower masking effect and opacity .

color of the underlying structure influenced the final

The color of PLV restorations might be affected bycolor of the PLV restoration.

30）

slight changes in the opacity and translucency . The
degree of translucency is directly related to the reflec30,31）

tion of light, and if the transparency decreases andthe surface reflects more light, the color of the PLV

Conflict of Interest
The authors declare no conflicts of interest associatedwith this manuscript.

tends to be whiter7,30）. Although compositional differences affect the transparency of resin composites30,32）,
using luting materials with different shade-matchingmechanisms does not affect TP values in PLV restorations within the limitations of this study.
In this study, the cement space was set at a thickness of 50μm versus a 2-mm underlying structure and1-mm PLV. The lack of difference in ΔE00 between the
different luting materials might have been because of
the narrow cement space relative to the overall thickness of the specimens, which might have had little
effect on the color. However, the ΔE00 value at Day 30
under all conditions exceeded the acceptable threshold,indicating a change in color. Tertiary amines are
responsible for the discoloration of dual-cure resin
cements over time10,11）, but in the present study a lightcured resin composite was used as the resin cement. In
addition, a light-cured resin composite was used as astandardized underlying structure instead of humanteeth to examine the effect of color. Therefore, there isa possibility that the underlying structure itself was
discolored by water. The effect of discoloration of theunderlying structure on the overall color tone cannot bedenied, which is a limitation of the present study. Different base resins of resin composites have different
quantities of unreacted monomers and water absorbency, and thus the effect of immersion in water on thecolor has been reported, especially a＊ and b＊32）. The
present study also indicated that the values of a＊ andb＊ changed significantly over time. Therefore, it is possible that the color change indicates a change in photo-polymerization resin composite although not as muchas in the chemical polymerization resin composite.

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Dec, 2023

113

Original Article

ODEP 3（1）：113～120，2023

Comparison of Root Canal Preparation Ability of Three Gold Wire-based
NiTi Rotary File Systems in Simulated Curved Root Canals
Miki SEKIYA, Shuntaro NAKAYAMA＊, Fumiyasu MURAYAMA, Kentaro FURUTA,
Yoshiyuki INUYAMA, Munehiro MAEDA, Kazuo KITAMURA＊ and Masaru IGARASHI
Department of Endodontics, The Nippon Dental University School of Life Dentistry at Tokyo＊

Division of General Dentistry 1（Endodontics）, The Nippon Dental University Hospital

Abstract
Purpose: The purpose of this study was to evaluate the effects of the ProTaper Ultimate（PTU）, which has
a new design based on integrated technology, on preparation ability in simulated curved root canals compared with previous gold wire-based nickel titanium rotary file systems.
Methods: The surfaces of J-shaped simulated root canals in clear resin blocks were scanned as image files.
The working length was defined as the root canal length minus 1 mm. The negotiation of all root canals was
confirmed with a stainless steel #10 K-file. The blocks were divided into three groups of 10 each（PTU, ProTaper Gold［PTG］, and WaveOne Gold［WOG］）and randomly prepared through size #25. The inner andouter displacements of the root canal wall were measured from superimposed images before and after
preparation. In addition, the working times of each group and process were measured. The Kruskal-Wallis
test followed by the Steel-Dwass test were used for all comparisons and statistical analyses.
Results: Regarding root canal wall displacement, no significant difference was observed in all groups at 1mm and 2 mm from the root canal apex, except for 1 mm on the inner side of the curvature（p＜0.05）. At 3mm and 4 mm from the root canal apex, the root canal wall displacement of PTU was significantly smaller
than those of PTG and WOG in the inner side of the curvature, while it was significantly larger in the outerside of the curvature（p＜0.05）. PTU prepared the inner and outer sides most evenly among the threegroups. The total working time and the root canal wall shaping time in each group were longer in the orderof WOG＜PTU＜PTG. Both times were significantly different among the three groups（p＜0.01）. In addition,
PTU had the smallest variance and preparation took almost the same amount of time even when the experiment was performed multiple times. On the other hand, there was no significant difference between groups
in the time required for glide path preparation.
Conclusion: The curved root canals at the measurement points were prepared most evenly by PTU in thisstudy. Also, the total working time and the root canal wall shaping time were significantly reduced with
PTU compared to PTG, and both times of PTU were close to the measurement time of WOG.
Key words: ProTaper Ultimate, ProTaper Gold, WaveOne Gold, nickel titanium instruments

Corresponding author: Miki SEKIYA, Department of Endodontics, The Nippon Dental University School of Life Dentistry atTokyo, 1‒9‒20, Fujimi, Chiyoda-ku, Tokyo 102‒8159, Japan
TEL: ＋81‒3‒3261‒5698, FAX: ＋81‒3‒5216‒3718, E-mail: sekiya-m@tky.ndu.ac.jp
Received for Publication: September 15, 2023/Accepted for Publication: November 13, 2023
DOI: 10.11471/odep.2023‒013

ODEP Vol. 3, No. 1

114

time during root canal preparation of curved root
canals.

Introduction

Given this background, the purpose of the present
The instruments and procedures used for root canalstudy was to evaluate the effects of PTU on prepara-treatment have changed substantially over time. In par-tion ability in simulated curved root canals comparedticular, the development and improvement of nickel

with previous gold wire-based NiTi rotary file systems

1）

titanium（NiTi）rotary files has been remarkable . It isby comparing the displacement of the inner and outer

now well-known that NiTi rotary files are more flexible

curvatures of the root canal wall and the working timethan conventional stainless steel files and have a higherof the file used for each group and process.

ability to follow curved root canals. The current NiTi
rotary files have enabled root canal preparation that

Materials and Methods

maintains the anatomical shape with a minimal amountof cutting, resulting in the establishment of a simple filesystem.

1 ．Preparation materials
Thirty J-shaped simulated root canals in clear resin

NiTi rotary files are becoming the gold standard ofblocks（root canal length: 16 mm, 0.02 taper, Endoroot canal preparation instruments, replacing handTraining Bloc J-Shape φ15, Dentsply Sirona）were usedfiles2‒5）. One of the factors that has greatly improvedin this study. The working length was unified to 15 mm,

the characteristics of NiTi rotary files in particular iswhich was 1 mm inside the apical foramen.

the strengthening of NiTi alloy by various heat treat-

The preparation groups were defined as PTU, PTG,

ment methods, which have enhanced the material prop-and WaveOne Gold（WOG）（Dentsply Sirona）, which

erties of many types of NiTi alloys6）. Heat treatment

are the three types of file systems based mainly on gold

technology has greatly increased the resistance towire.

repeated fatigue, improved the flexibility of the file, andmade it possible to design the file shape to optimize
shaping performance. NiTi rotary files continue to be
developed with this technology3‒5,7）.

2 ．Preparation of J-shaped simulated root canals
in clear resin blocks
All blocks were divided into three groups of 10 each,and negotiation was confirmed with a #10 K stainless

Since its introduction in 2001, the ProTaper systemsteel file（SSK）（READYSTEEL FlexoFile; Dentsply（Dentsply Sirona, Ballaigues, Switzerland）has under-

Sirona）. Next, the model blocks were prepared throughgone constant evolution and improvements owing tosize #25 according to the operation method specified byadvances in technology and accompanying unique filethe manufacturer in each system-specific mode pro-

designs . In particular, ProTaper NEXT using M-wiregrammed in the X-Smart IQ motor handpiece（Dentsply

and ProTaper Gold（PTG）using gold wire appeared

Sirona）. All root canal preparations were performed byafter the heat treatment of NiTi alloys began, followeda single operator with 6 years of clinical experience in aby the latest file system, ProTaper Ultimate（PTU）,

state in which the root canal morphology of the block

which inherited the characteristics of the previous Pro-was not visible. In addition, the order of root canal

8）9）Taper file system . Each PTU file was subjected topreparation was randomly selected in each group. To

individual heat treatment and design to maximize theachieve the same conditions as the effect of root canalperformance of each of its roles, and all heat treatment

irrigants on files in clinical practice, frequent root canaltechnology and file designs were integrated within theirrigation with 3％ sodium hypochlorite solution

system.

（NaOCl）（amplitude, 2‒3 mm）
was commonly performed

The method for evaluating the displacement of theusing a syringe（2.5 mL; Terumo, Tokyo, Japan）androot canal wall when preparing a curved root canal isirrigation needle（30 G; Dentsply Sirona）during root

often used as a means to clarify the cutting efficiency of

canal preparation. In addition, the files were cleaned of

NiTi rotary files. However, to the best of our knowl-

debris and the root canals recapitulated with a #10 SSK

edge, no studies have compared PTU and previous gold

after each time.

wire-based NiTi rotary file systems in terms of the

PTU was used with all files（400 rpm and 4.0 Ncm

amount of root canal wall displacement and working

torque）. After securing straight-line access with an SX

Dec, 2023

Root Canal Preparation Ability of Gold Wire-based NiTi Rotary Systems

115（20/.03）, glide path preparation was performed using
the Slider（16/.02）. In the presence of viscous chelating
agents（15％ EDTA; Glyde, Dentsply Sirona）, the Slider
instrument reached the apical side once with gentle
up-and-down movements. Next, under 3％ NaOCl infiltration, a Shaper（20/.04）shaping file was used in a

Inner

gentle brushing motion until the working length was

Outerreached. After that, the finishing files were used up to
4 mm
3 mm
2 mm
1 mm

the working length by moving up and down in theorder of F1（20/.07）and F2（25/.08）. The files inserted
to the working length were not left for more than one
second and immediately removed from the root canal tocomplete the preparation.
The number of PTG revolutions was unified at 300

0 mm（apex）
Fig. 1rpm. SX and S1 were used with a torque value of 4.0

Measurement method for root
canal wall displacement

Ncm, ProGlider with 2.0 Ncm, S2 and F1 with 1.5 Ncm,
and F2 with 3.0 Ncm. After straight-line access to thethat the blocks could always be scanned at the sameroot canal orifice had been created with an SX（19/.04）

position. After root canal preparation, the blocks werein the presence of 15％ EDTA, the glide path was pre-scanned again and superimposed with the images

pared with ProGlider（16/.02）. The S1（18/.02）and S2scanned beforehand using Photoshop CS6（Adobe, San（20/.04）shaping files were prepared in order along theJose, CA, USA）
. The measurement points of the root

glide path under 3％ NaOCl irrigation until the working

canal wall displacement were set to 1 mm, 2 mm, 3 mm,

length was reached. At that time, a brushing motion

and 4 mm from the root canal apex. The measurement

was performed on the root canal wall until light resis-

method followed that of Yun et al.10）. A line perpendicu-

tance was felt. The F1（20/.07）and F2（25/.08）finish-

lar to the long axis of the root canal was drawn at eaching files were inserted deep into the root canal by verti-

measurement point, and the amount of root canal wall

cal movement until reaching the working length. All

displacement（the distance of the root canal wall before

files inserted to the working length were then immedi-

and after preparation）was measured in the inner andately withdrawn from the root canal to complete theouter sides of the curvature（Fig. 1）.

root canal preparation.

Measurement of the root canal preparation time wasRegarding WOG, straight-line access was preparedstarted after confirmation of the negotiation by the SSK

using the PTG SX, and then a WOG Glider（15/.02）wasand completion of straight-line access to the root canalused to prepare the glide path up to the working lengthorifice with the SX file in each file system. The total

with a gentle up-and-down reciprocating motion. Next,

time needed to complete the preparation for each groupprimary（25/.07）weak pecking motions were repeated

was calculated by subtracting the time required for

two or three times to shape to the coronal 1/3. Using

root canal irrigation, instrument exchange, cleaning of

the same procedure, root canal preparation was per-debris, and recapitulation from the measured time until

formed to the crown side 2/3 and then to the working

the end of root canal preparation. Using the samelength. As soon as the working length was reached, themethod, the time required for glide path preparation

file was removed from the root canal to complete theand subsequent root canal shaping in each group wasroot canal preparation.

also calculated.

3 ．Evaluation of simulated root canal preparations

4 ．Statistical analysis
All values were expressed as mean±standard devia-

Before the root canal preparations, the curved sur-tion. SPSS Statistics（version 28; IBM Japan, Tokyo,

face of each block was scanned as an image file using a

Japan）and Kyplot 6.0（KyensLab, Tokyo, Japan）were

flatbed color image scanner（1200 dpi, GT-X970; EPSON,

used for the statistical analysis, with the significanceTokyo, Japan）. The positional conditions were unified solevel set at 5％.

ODEP Vol. 3, No. 1

116
Table 1

Root canal wall displacement at each measurement point in the inner and outer sides of the curvature in each group（mm）（mean±standard deviation）

The measurement
points from apex
4 mm

ProTaper Ultimate
Inner

Outer

ProTaper Gold
Inner

＊＊

3 mm

0.17±0.01
0.15±0.02

0.13±0.01
0.12±0.01＊＊

2 mm

0.11±0.02

0.10±0.01＊＊

1 mm

0.07±0.01

0.08±0.01＊＊

WaveOne GoldOuter

0.23±0.02
0.19±0.02＊＊

0.12±0.02
0.10±0.01＊＊

0.12±0.02

0.10±0.01＊＊

0.09±0.01

0.08±0.01

＊＊

Inner

Outer

0.21±0.03
0.17±0.02

0.12±0.01＊＊
0.11±0.01＊＊

0.11±0.02
0.08±0.01

0.10±0.01＊＊
0.08±0.01＊

Statistically significant differences were observed between the inner and outer sides of the curvature ineach group（Wilcoxon signed-rank test, ＊p＜0.05, ＊＊p＜0.01）

Normality was confirmed by the Shapiro-Wilk test,
followed by one-way analysis of variance（ANOVA）if
normality was observed, and the Kruskal-Wallis test if

2 ．Measurement of simulated root canal preparation time
The measurement times in each group and for eachnot. The post-hoc test after one-way ANOVA was per-process are shown in Fig. 3, and the times needed toformed by Tukey’s test or the Games-Howell testcomplete each file system’s procedure and each file are

depending on the results of Levene’s test for equality ofshown in Fig. 4.

variances. In addition, the Steel-Dwass test was used
for the post-hoc test after the Kruskal-Wallis test.

The total time needed to complete preparation ineach group was longer in the order of WOG（27.05±
4.87 s）＜PTU（32.20±1.66 s）＜PTG（50.01±5.24 s）, and

Results

significant differences were observed between allgroups（p＜0.01）. On the other hand, the time required

In this study, no file fractures, perforations, apicalfor glide path preparation was 6.55±1.27 s, 6.55±1.27 s,

blockages, or loss of working length were observed inand 6.26±1.39 s in PTU, PTG, and WOG, respectively,

any group.

with no significant differences observed between any

1 ．Simulated root canal wall displacement

groups. The root canal shaping time of each group was

The root canal wall displacement at each measure-

longer in the order of WOG（20.79±0.69 s）＜PTU（25.99

ment point on the inner and outer sides of the curva-±0.26 s）＜PTG（43.46±0.89 s）, with significant differ-ture in each group is shown in Table 1. At 1 mm and 2ences observed between all groups（p＜0.01）.

mm from the root canal apex, the displacement of theinner and outer sides of the curvature were almost

Discussion

equal in each group. At 3 mm and 4 mm from the root
canal apex, PTG and WOG were displaced to the inner

Ideal root canal preparation involves minimizing theside of the curvature. PTU had the most equal displace-transposition of the root canal after preparation andment of the inner and outer sides of the curvature at 2

providing a continuous taper from the root canal orifice

mm, 3 mm, and 4 mm from the root canal apex.

to the apex while preserving the original root canal

The comparison of root canal wall displacement inmorphology11‒13）. Especially in the case of curved root

each group is shown in Fig. 2. At 1 mm and 2 mm from

canals, it has been reported that the success rate of

the root canal apex, significant differences wereroot canal treatment is 78.9％ if the curvature can beobserved between PTU and PTG and between PTG

followed, and 42.9％ if it cannot14）. Furthermore, inand WOG only on the 1 mm inner side of the curvaturerecent years, emphasis has been placed on minimizing（p＜0.01）. At 3 mm and 4 mm from the root canal apex,

the amount of root canal dentin removed, based on thethe root canal wall displacement of PTU was signifi-

15‒17）
concept of minimal intervention（MI）.

cantly smaller than PTG and WOG in the inner side of

In this study, we compared the amount of root canalthe curvature, while it was significantly larger in thewall displacement and working time during root canal

outer side of the curvature（p＜0.05）.

preparation of simulated curved root canals using theDec, 2023

Root Canal Preparation Ability of Gold Wire-based NiTi Rotary Systems

117

Inner
Outer
Measurement points from canal apex
4mm

3mm

2mm
ProTaper Ultimate
ProTaper Gold1mm（mm）0.30

0.25

0.20

0.15

0.10

0.05

0

WaveOne Gold
0

0.05

0.10

0.15

0.20

0.25

0.30（mm）

Root canal wall displacement at each measurement point in the inner and outer sidesof the curvature in each group
Kruskal-Wallis test followed by Steel-Dwass test, ＊p＜0.05；＊＊p＜0.01.

Fig. 2（A）（B）

ProTaper Ultimate

ProTaper Ultimate

ProTaper Gold

ProTaper Gold

WaveOne Gold

WaveOne Gold0

10 20 30 40 50 60（S）

0

10 20 30 40 50 60（S）

0

2

4

6

8

10（S）

（C）
ProTaper Ultimate
ProTaper Gold
WaveOne Gold

Fig. 3 Measurement time in each group and for each process
Kruskal-Wallis test followed by Steel-Dwass test, ＊p＜0.05；＊＊p＜0.01（A）Total time needed to complete preparation,（B）Glide path preparation time,（C）Root
canal shaping time

ProTaper system and the WaveOne system, based onvents the file from over-engagement and releases

gold wire. The ProTaper system performs root canalexcessive torque through a reciprocating motion based

preparation using the full-length method in which multi-

on Roane’s balanced force technique to avoid straight-

ple files have different roles. In addition, multiple tapers

ening of the root canal curvature18）. Therefore, a sin-

are added to one file. As a result, a simpler system with

gle-file system was established and simple root canalfewer files than the conventional NiTi rotary file sys-

preparation was realized in a short time.

8）

tem has been realized . The WaveOne system pre-Regarding root canal preparation in curved root

ODEP Vol. 3, No. 1

118

ProTaper Ultimate Slider

Shaper

ProTaper Gold ProGlider

S1

WaveOneWaveOne Gold GoldGlider

Primary0
Fig. 4

10

F1

F2
S2

20

F1

30

F2

40

50（S）

Measurement time needed to complete each file system’s procedure

canals, ideally the displacement of the root canal wallgroup at 1 mm and 2 mm from the root canal apex.

should be in the ratio of inner side to outer side＝1：1

Therefore, it was suggested that all file systems had（inner side/outer side＝1）11）. In this study, PTU had

good followability near the root canal apex.

the most equal displacement of the inner and outer

The total working time and root canal wall shaping

sides of the curvature at 2 mm, 3 mm, and 4 mm fromtime in each group were longer in the order of WOG＜

the root canal apex, which was the closest to the ideal

PTU＜PTG. This result was consistent with thatroot canal preparation morphology. This result indi-

reported by Bürklein et al. 22）and Bane et al.23）, whocated that the central axis of PTU was close to the cen-compared the time required for root canal preparation

tral axis of the root canal. Many studies have shownusing multi- and single-file systems. Accordingly, it wasthat more flexible instruments produce more centeredshown that the time needed to complete each file sys-

root canal preparations

19,20）. Therefore, PTU was con-tem’s procedure was affected by the number of files

sidered to have higher flexibility and ability to follow

used to shape the root canal. In addition, PTU had thethe root canal up to the apex than other file systems

smallest variance, which indicates that the PTU system

due to its unique file diameter and cross-sectional

has little variation in technique and can be prepared indesign. However, at 1 mm and 2 mm on the apical side,

the same amount of time, even if it is used multiple

a significant difference was observed between PTG andtimes. Comparing the time needed to complete each fileother systems only on the inner side of the curvaturesystem’s procedure, PTU and PTG, which are multi-fileat 1 mm. This result was consistent with the report bysystems, had a shorter operating time per file thanShi et al. who studied the centering ability of PTG and

WOG, the single-file system. Therefore, it was thought

21）. In addition, this result was thought to bethat PTU and PTG can be reused after sterilization

affected not only by the file cross-sectional design andbecause fatigue is less likely to accumulate in files com-

the file taper, but also by the number of times the filepared with WOG, which is for single use.

WOG

reached the working length. PTG is a full-length

On the other hand, no significant difference in themethod and multi-file system. In addition, in the present

time required for glide path preparation was seen

study, the PTG system had the largest number of files

between any groups. As one of the precautions whenused in the root canal preparation protocol. Therefore,

using NiTi rotary file systems, almost all manufacturers

in PTG, it seems that the displacement occurredcurrently recommend glide path preparation, which is

because of the file reaching the working length multiple

an essential step for the safe and effective use of NiTitimes. In particular, the NiTi rotary file systems used inrotary systems24‒27）. Cassim et al.28）reported that glide

this study were mainly made of highly flexible gold

path preparation reduced the risk of torsional stresswire. Thus, when inserted into a curved root canal, all

and instrument fracture and maintained the original

files follow the inner curved wall without causing

root canal anatomy with less modification of the root

springback, which explains why there was a significant

canal curvature and fewer root canal aberrations. In

difference only on the inner side of the curvature in thisaddition, Keskin et al.29）reported that glide path prepa-

study. However, the displacement of the inner andration improved the shaping ability of NiTi files. It hasouter sides of the curvature were almost equal in eachbeen stated that glide path preparation using hand files

Dec, 2023

Root Canal Preparation Ability of Gold Wire-based NiTi Rotary Systems

119

should be used from #10 to #15 24,25）or from #10 toConflict of Interest#2030‒32）. However, glide path preparation using hand

The authors declare no conflict of interest related to thispaper.

files takes a long time and involves the risk of transportation from the root canal morphology at the apex. The
NiTi rotary files for glide path preparation can establish the glide path in a shorter time than hand files33）.
Regarding glide path preparation files, Slider and ProGlider used in a continuous rotational motion are madeof M-wire, and the WOG Glider used in a reciprocating
motion is made of gold wire. Gold wire has lower cutting force than M-wire due to the higher proportion ofmartensitic phase. However, it is thought that thereciprocating motion freed the narrow root canal fromthe torsional fractures that tend to occur with small
files, and enabled efficient glide path preparation to theroot canal apex. Therefore, in addition to improving
machinability and root canal followability through heat
treatment technology, by combining a file rotation
method according to the properties of the NiTi alloy, itis thought that glide path preparation in a short time ispossible using any file system.
These results suggest that root canal preparation by
PTU was closest to the ideal curved root canal preparation that avoids excessive enlargement based on the
MI concept and provides excellent root canal followability, with working time closer to WOG than PTG. In
addition, we considered that all file systems based ongold wire had good followability near the root canal
apex and reduced the risk of inappropriate root canal
preparation.

Conclusion
As a result of this study, the following conclusions
were reached:
1．The curved root canals at the measurement
points were prepared most evenly by PTU.
2．All gold wire-based NiTi rotary file systems usedin this study had good followability near the root canal
apex.
3．The total working time and the root canal wall
shaping time were significantly reduced with PTU
compared to the previous PTG system, and both timesof PTU were close to the measurement time of the
WOG single-file system.
4．All NiTi rotary files used for glide path preparationin this study achieved smooth performance in a short time.

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Dec, 2023

121

Original Article

ODEP 3（1）：121～128，2023

2-deoxy-D-glucose Inhibits Cell Activity by Inhibiting Intracellular Glucose
Metabolism in Human Gingival Fibroblasts
Yoichiro TAGUCHI, Hirohito KATO, Runbo LI, Tsurayuki TAKAHASHI, Daisuke KIMURA,
Kosho KASHITANI, Niina MASU, Hitoshi AZUMA, Chizuko OGATA and Makoto UMEDA
Department of Periodontology, Osaka Dental University

Abstract
Purpose: Cellular energy metabolism by glucose plays an essential role in tissue wound healing. The use of
2-deoxy-D-glucose（2-DG）to suppress energy metabolism through the glycolytic system is similar to a low
glucose environment. Therefore, the purpose of this study was to investigate the effect of 2-DG-induced
reduction in glucose metabolism on cell viability.
Methods: We examined the proliferation, migration, cytotoxicity, H2O2 production, glucose transporter type1 expression, lactic acid production, and ATP synthesis after the intracellular uptake of three different concentrations of 2-DG into HGnFs.
Results: 2-DG suppressed intracellular glucose metabolism, which led to a reduction in the cellular activityof human gingival fibroblasts（HGnFs）. However, there were no changes in cytotoxicity or H2O2 production,and the effects on glucose metabolism were different from those in a low glucose environment.
Conclusion: This study showed that the suppression of intracellular glucose metabolism in HGnFs by 2-DG
may delay wound healing in periodontal tissues.
Key words: glucose, 2-DG, gingival fibroblasts

Corresponding author: Yoichiro TAGUCHI, Department of Periodontology, Osaka Dental University, 8‒1, Kuzuha-hanazono-cho, Hirakata, Osaka 573‒1121, Japan
TEL: ＋81‒72‒864‒3084, FAX: ＋81‒72‒864‒3184, E-mail: taguchi@cc.osaka-dent.ac.jp
Received for Publication: September 15, 2023/Accepted for Publication: November 16, 2023
DOI: 10.11471/odep.2023‒014

ODEP Vol. 3, No. 1

122cannot be generated because of the lack of glucose8）.
In our previous study, we reported the impact of low

Introduction

glucose levels on the proliferation and migration of
Human gingival fibroblasts（HGnFs）are the most

HGnFs, primarily attributable to their inability to gen-abundant cellular component of periodontal tissue anderate energy9）. Building upon this premise, our current

play an important role in the wound healing of gingival

investigation delves into the behavior of HGnFs, encom-

1）and oral mucosal tissues . During wound healing,

passing their proliferation and migration, through the

HGnFs produce various growth factors and are actively

lens of wound healing. This is achieved by employing

involved in the formation of granulation tissues, espe-

2-DG to artificially create a low glucose environment,

cially the synthesis of collagen. Collagen is a complex

mirroring situations where HGnFs face energy produc-

extracellular matrix that is important for the regenera-tion challenges.

2）

tion of functional periodontal tissue . Wound healing

The results of this study unveil how 2-DG influences

depends on collagen synthesis, which is directly depen-cellular energy metabolism, effectively mirroring condi-

dent on the proliferative and migratory capacity oftions characterized by low glucose concentrations.

HGnFs 3）. Thus, the proliferation and migration of

Insights drawn from this study have important implica-

HGnFs are considered the most important factors oftions for both future research and clinical applications.

wound healing.

By shedding light on how cells respond to low glucose

HGnFs utilize the glycolytic pathway to promote

environments, this research paves the way for a deeper

their proliferation and migration4）. The role of glucose

understanding of cellular dynamics in settings marked

in cell proliferation is highly dependent on glucose

by nutrient insufficiency. These insights could be lever-

metabolism, which provides a source of energy as well

aged in future studies to explore novel therapeutic

as metabolites for the biosynthesis of nucleic acids andapproaches or interventions targeting cells struggling

5）

membrane lipids . However, when the existing blood

with energy deficits, thereby enhancing their prospects

vascular system is disrupted by surgery or other treat-for clinical application in conditions related to glucose

ments or trauma, cells are exposed to a stressful micro-

metabolism irregularities.

environment characterized by low glucose concentrations. Furthermore, the blood supply in periodontal tis-

Materials and Methods

sues after trauma or periodontal surgery is significantly
lower than that in healthy periodontal tissues, and periodontal tissues are exposed to a low glucose concentra-

1 ．Cell culture and culture conditions with 2-DG
solutiontion environment. Previous studies have been con-

HGnFs were obtained from ScienCell Research Labo-ducted under various glucose conditions to mimic theratories（San Diego, CA, USA）
. HGnFs were incubatedhigh glucose concentration model of the diabetic state.

in Dulbecco’s modified eagle medium（DMEM）（Nacalai

We have also conducted several previous studies that

Tesque, Kyoto, Japan）with a normal glucose concentra-

mimicked various clinical environments by increasingtion（100 mg/dL）, and one of three 2-DG concentrations

6）

or decreasing glucose concentrations . However, few（100, 500, 1,000μM）.

studies have investigated cell behavior under low glu-

2 ．Cell proliferation assay

cose concentrations that mimic the environment of an

HGnFs were plated in 96-well microplates at 1×104

inadequate nutrient supply. A low glucose environment

cells/mL in a 100μL culture medium. The cells were

can be obtained using 2-deoxy-D-glucose（2-DG）or bycultured to adhere for 24 h, and then the medium wasintentional glucose reduction. 2-DG, a glucose molecule

exchanged to a medium with different 2-DG concentra-

in which the 2-hydroxyl group is replaced by a hydro-tions（100, 500, 1,000μM）. Proliferation data were deter-

gen atom, is not metabolized by the glycolytic system.

mined using Cell Count Reagent SF（Nacalai Tesque）

How 2-DG inhibits cell growth is not entirely clear, but

and analyzed using SoftMax Pro Microplate Data

it is taken up by cellular glucose transporters, similar to

Acquisition and Analysis software（Version 7.0, Molec-

7）

glucose , but it is not metabolized by the glycolytic
system, analogous to an environment where energy

ular Devices, Sunnyvale, CA, USA）.
For live and dead staining, HGnFs were stained with

Dec, 2023

2-DG Affects Cell Activity of Human Gingival Fibroblasts

123

calcein-acetoxymethylester（calcein-AM）and propidium

6 ．Gene expression in HGnFs

iodide（PI）solutions, respectively（Dojindo, Kumamoto,Total RNA was isolated using an RNeasy Mini Kit

Japan）. Images of the stained cells were obtained using（Qiagen, Venlo, Netherlands）. RNA from each sample

a BZ-Ⅱ all-in-one fluorescence microscope（Keyence

was reverse-transcribed to cDNA using a PrimeScript

Corporation, Osaka, Japan）.

Reagent Kit（Takara Bio Inc., Shiga, Japan）. The gene

3 ．Wound-healing assay

expression of GLUT1（TaqMan Gene Expression Assay;

Wound-healing assays were performed using a

Applied Biosystems, Thermo Fisher Scientific）waswound-repair assay kit（Ibidi GmbH, Martinsried, Ger-

quantified using the StepOnePlus Real-Time PCR Sys-

many）following the manufacturer’s instructions.

tem（Thermo Fisher Scientific）.

Briefly, HGnFs were plated at 3.5×104 cells/70μL in a

7 ．Measurement of lactic acid production andcell culture insert. When the cells reached complete

ATP levels

confluence, the culture insert was lifted and theLactic acid production was measured using a Lactate

medium was replaced with a serum-free medium with

Assay Kit-WST（Dojindo）according to the manufac-

different 2-DG concentrations（100, 500, 1,000μM）.

turer’s instructions. HGnFs were stimulated with dif-Then, images of each wound at 0 and 24 h were photo-

ferent concentration of 2-DG（100, 500, 1,000μM）, thegraphed and analyzed.

supernatants collected for analysis, and the data nor-

4 ．Cytotoxicity assay and Reactive Oxygen Spe-malized to DNA. ATP levels were determined using
the CellTiter-Glo 2.0 luminescent cell viability assay

cies（ROS）detection
Lactate dehydrogenase（LDH）activity was measured（Promega, Madison, WI, USA）. Briefly, 1×104 cells/

using the Cell Cytotoxicity LDH Assay Kit-WST kit

100μL were loaded into each well in a 96-well plate.

（Dojindo）, following the manufacturer’s instructions.

The cells were cultured to adhere for 24 h, and then4

the medium was exchanged to a medium with different

cells/mL in 100μL culture medium. The cells were cul-

2-DG concentrations. Then, the cells were incubated fortured to adhere for 24 h, and then the medium was24, 48, 72, or 120 h. After the addition of 100μL volume

exchanged to a medium with different 2-DG concentra-of CellTiter-Glo reagents, relative luminescence units

tions（100, 500, 1,000μM）and incubated for 24 h. The

were measured following the manufacturer’s instruc-absorbance of each well was measured at 490 nm. ROS

tions.

HGnFs were plated in 96-well microplates at 2×10levels were measured using a hydrogen peroxide fluorometric detection kit. Briefly, the culture supernatants

Resultsof HGnFs were collected and pipetted into a black
96-well microplate（Nunclon Delta Surface 96-well

1 ．Cell proliferation

microplate, Thermo Scientific, Waltham, MA, USA）and

Figure 1 shows the effects of various 2-DG concen-

incubated for 10 min at room temperature in the dark,

trations on HGnF proliferation after culturing for 24, 48,

and the fluorescence was measured at 570 nm exci-

72, and 120 h. The number of live cells stained withtation and 600 nm emission.

calcein-AM solution decreased proportionally at 72 and

5 ．Immunofluorescence staining

120 h. However, no detectable dead cells were foundFollowing 2-DG stimulation, cells were incubatedafter staining with PI solution, regardless of the concen-with primary antibodies against human type Ⅰ colla-

tration of 2-DG used. HGnF proliferation was signifi-gen（Abcam, Cambridge, UK）, glucose transporter typecantly increased by 100μM 2-DG for the first 72 h.

1（GLUT1）（Abcam）, and ATP5B（Santa Cruz Biotech-

Subsequently, 2-DG significantly suppressed the prolif-

nology, Inc., Dallas, TX, USA）
. Fluorescent immunos-

eration of HGnFs and decreased the number of live

taining was performed using Alexa Fluor 488R-conju-

cells stained with calcein-AM.

gated secondary antibodies（Thermo Fisher Scientific）

2 ．Wound healing capability

and nuclei were stained with 4’,6-diamidino-2-phenylin-

The wound healing capability of HGnFs was exam-

dole（DAPI）（Dojindo）. Images of stained cells wereined using a scratch assay. Figure 2 shows that 100μM

obtained by fluorescence microscopy. These images

2-DG significantly promoted wound healing. By con-were then analyzed.

trast, incubation with 1,000μM 2-DG significantly inhib-

ODEP Vol. 3, No. 1

124
0 μM

A

100 μM

500 μM

1,000 μM

B
Calcein

PI

PI

Merge

Merge

80
60
40
20
0

72 h
0 μM
100 μM
500 μM
1,000 μM

D

††

＊＊
††

††
††

††

72

120

（h）

100 μM0 μM
100 μM
500 μM
1,000 μM

1.0
0.5
0.0

500 μM

1,000 μM

120 h

1.5
Cell proliferationC

Calcein stained area（％）

Calcein

0 μM

††

＊＊
††
††

24

48

72

††
††

120 （h）

Fig. 1 Different 2-deoxy-D-glucose（2-DG）concentrations affect the proliferation of human gingival fibroblasts（HGnFs）（A and B）HGnFs stained by calcein-AM and PI were photographed under a fluorescence microscope after incubation
with 2-DG for 72 and 120 h.
（C）Viable and dead cell staining is presented as the percentage of the calcein-stained area.
（D）Cell proliferation was measured after incubation with 2-DG for 24, 48, 72, and 120 h. Scale bars：500μm. ＊＊：p＜0.01, significant increase compared with the control group；††：p＜0.01, significant decrease compared with the control
group.

ited cell migration.

shows that lactate production was significantly

3 ．Cytotoxity and cellular stressdecreased by 500 and 1,000μM at 72 h; however, at 120

Figure 3A shows that cytotoxicity was generally low

h, lactate production was partially decreased in all

in the 2-DG-stimulated groups. In addition, cytotoxicity

2-DG-stimulated groups. By contrast, lactate production

was reduced in the 1,000μM 2-DG group.

was significantly decreased in the 1,000μM group at 24,

Hydrogen peroxide（H2O2）, a marker of ROS, was72, and 120 h.

measured after culturing for 24, 72, and 120 h. Figure

The intracellular ATP levels were significantly

3B shows no significant change in H2O2 production inincreased by 100μM 2-DG at 72 h, and at 120 h, werethe 2-DG-stimulated groups, indicating a generalsignificantly decreased in all 2-DG-stimulated groups.

absence of cellular stress related to 2-DG stimulation.

The activity of ATP5B was improved by 100 and 500

4 ．Glucose uptake capability

μM 2-DG at 72 h, with the highest increase detected

Figure 4 shows that GLUT1 expression in HGnFs

for 100μM 2-DG. After 120 h, ATP5B activity in theincreased as the concentration of 2-DG was increased

2-DG-stimulated groups was significantly reduced com-as analyzed by immunofluorescence staining and real-pared with that in the 0μM 2-DG group.

time PCR. The expression of GLUT1 mRNA was significantly increased in the 500 and 1,000μM 2-DG

Discussion

groups after 72 h.
5 ．Glycolysis levels

This study investigated the effect of an environment

Lactate production and intracellular ATP levels

in which energy metabolism cannot be performed, on

under low glucose conditions were measured after cul-

HGnFs using 2-DG as an in vitro gingival wound heal-

turing HGnFs with 2-DG for 24, 72, and 120 h. Figure 5

ing model. Our results showed that different concentra-Dec, 2023

2-DG Affects Cell Activity of Human Gingival Fibroblasts

A

125

B
100 μM

500 μM

1,000 μM

60
Wound closure（％）

0 μM
0h

24 h

＊

40
††

20
0

0

100

500 1,000（μM）

Fig. 2 Different 2-DG concentrations affect the wound-healing capability of HGnFs（A）The process of wound healing assay was photographed at 0 and 24 h. Scale bars：500μm.（B）The data of thewound-healing assay are presented as the ratio of the final scratch size to initial scratch size. ＊：p＜0.05, significant
increase compared with the control group；††：p＜0.01, significant decrease compared with the control group.

B

Cytotoxicity
（％）

100
80
60
40
20
0

†

0

100

500 1,000（μM）（mM）
H2O2 concentration

A

15
10

0 μM
100 μM
500 μM
1,000 μM

5
0

24

72

120

（h）

Fig. 3 The addition of 2-DG did not affect the cellular stress levels of HGnFs（A）Cytotoxicity（LDH level）was determined after 24 h of incubation with 2-DG.（B）The
level of ROS was examined using a hydrogen peroxide（H2O2）detection kit. After 24, 72,

and 120 h of incubation with 2-DG, the supernatant was collected and the level of H2O2 wasmeasured by a microplate reader. †：p＜0.05, significant decrease compared with the control group.

tions of 2-DG induced different cellular functions, suchthe low glucose environment induced by 2-DG, and theyas cell proliferation and migration. These results are

initiate energy production. In contrast, at later time

similar to our previous study on the effect of low glu-

points, it is conjectured that the inhibition of glucose

9）

cose environments on HGnFs . The optimal concentra-

metabolism caused by 2-DG suppressed the cell prolif-tion of 2-DG was not determined in this study. In theeration. Many studies have reported that 2-DG sup-present study, different concentrations of 2-DG werepressed the proliferation and migration of cancer

added to inhibit cellular glucose metabolism to different

cells10‒12）. Our previous results showed that a low glu-

degrees, and experiments were carried out at threecose environment suppressed the proliferation andconcentrations, as the aim was to investigate the biolog-

migration of HGnFs and affected their cellular activi-

ical changes in the cells.

ties. The present results suggest that 2-DG suppresses

In Fig. 1, the data show that 2-DG did not alter
HGnFs’ cell proliferation at 48 h. However, a significant

the proliferation and migration of HGnFs by reducing
their energy metabolism.

inhibition of cell proliferation by 2-DG was observed at

In the present study, no cytotoxicity or production of

72 and 120 h. This early increase in proliferation likely

H2O2, a reactive oxygen species, was observed whencorresponds to the phase in which cells are adapting to

the energy metabolism of HGnFs was inhibited by

ODEP Vol. 3, No. 1

126

0 μM

100 μM

GLUT 1500 μM

1,000 μM

B
Glut 1 mRNA expression

A

2.5
＊＊

2.0
1.5

＊＊

＊

1.0
0.5
0.0

0

100

500

1,000（μM）

Fig. 4 2-DG increases Glut 1 expression in HGnFs（A）HGnFs were photographed by a fluorescence microscope after incubation with a fluorescently labeled secondary antibody. Nuclei were stained with DAPI.（B）The gene expression of GLUT1 mRNA was determined by qPCR of stimulated HGnFs incubated for 72 h.
Scale bars：100μm. ＊：p＜0.05, ＊＊：p＜0.01, significant increase compared with thecontrol group.

2-DG. Our previous studies showed that cytotoxicity

cose uptake and glycolysis. During glucose uptake,and the production of H2O2 and ROS increased withtransportation across the cellular membrane is thedecreasing glucose concentrations under a low glucose

main rate-limiting step, which is determined by theenvironment. Cytotoxicity and H2O2 production differ

facilitated diffusion of GLUTs13）. GLUT1 is responsible

between low glucose environments and the in vitro

for the low level of basal glucose uptake required toenvironment induced by 2-DG in this study. Vibhuti etmaintain respiration in all cells14）. In the present study,

al. reported that the cytotoxicity of HGnFs and produc-a concentration-dependent increase in the expression oftion of ROS increased with increasing concentrations of

GLUT1 was observed when HGnFs were cultured with

13）

2-DG . In Fig. 3, no ROS formation was observed fol-

2-DG. We reported that low glucose enhanced thelowing 2-DG treatment. This observation suggests that

expression of GLUT1 in HGnFs, which suggests that

2-DG may diminish cellular glucose metabolism and,low glucose might enhance glucose uptake proportion-consequently, decrease the oxidative phosphorylation

ally as HGnFs sense glucose deprivation8）. Further-process. Under conditions of low glucose, there is typi-

more, the elevated expression of GLUT1 occurs in envi-

cally a reduction in oxidative phosphorylation within theronments where there is reduced cellular metabolism

mitochondria, leading to a decrease in ROS production.

via the glycolytic pathway.

Therefore, the decrease in glucose metabolism induced

Glycolysis is a complex biochemical process mediatedby 2-DG is likely to contribute to the suppression ofby multiple glycolytic genes and pathways. Lactate is

ROS production. This aligns with the absence of cellular

an intermediate cellular metabolite of the glycolytic

stress, indicating that 2-DG may exert relatively safe

pathway and is produced in the cytoplasm through theeffects on cells. We previously reported that a low glu-

reduction of pyruvate15）, and ATP is the end metabolitecose environment was detrimental to HGnFs, by induc-of glycolysis16）. Thus, their levels may reflect the level

ing inflammatory cytokine expression and cellularof glycolytic activity of a cell17‒19）. Our results14）showed

14）

stress . These results are similar to the low glucosethat lactate and ATP production were relatively

environment and the in vitro environment using 2-DG

increased in the 100μM 2-DG group at 72 h, whichin the present study, whereby energy production ismight indicate enhanced glycolytic activity. However,reduced because of decreased cellular metabolism via

there was a decrease in lactate and ATP production

the glycolytic system. However, in terms of the cyto-

compared with that in the control group at 120 h as the

toxicity of HGnFs, it may be different.

2-DG concentration was upregulated, similar to our pre-Glucose metabolism consists of two processes: glu-

vious studies using low glucose environments. This

A

2-DG Affects Cell Activity of Human Gingival Fibroblasts

Lactate（mM）

3

B

0 μM
100 μM2††500 μM
1,000 μM

††

††

††

††

1
††

0

C

24

0 μM

72

120

（h）

100 μM

ATP Luminescence（RLU）

Dec, 2023（×106）
1.5
1.0

127

0 μM
100 μM††

500 μM
1,000 μM

0.5
0

††
††

24

††

††

††

72

500 μM120（h）1,000 μM

ATP5B

Fig. 5 The addition of 2-DG affects the glycolysis levels in HGnFs（A）
Lactate production by HGnFs exposed to 2-DG was measured after incubation for 24, 72, and 120 h.（B）Thelevel of intracellular ATP was determined by luminescence using a microplate reader after incubation for 24, 72,
and 120 h.（C）The activity of ATP5B was photographed by a fluorescence microscope after incubation for 120 h.
Scale bars：100μm. ††：p＜0.01, significant decrease compared with the control group.

result indicated that glucose metabolism in the 100μM

between in vivo low glucose environments and a low

2-DG group was higher than that in the control group inglucose environment induced by 2-DG incorporation.

the first 72 h, which might explain the enhanced woundhealing capacity of the 100μM 2-DG group at 72 h. At

Conclusion

120 h, glucose metabolism was reduced in all 2-DG
groups, which might indicate that glucose uptake was

The results of this study suggest that the intracellu-

not sufficient to support glucose consumption levels

lar uptake of 2-DG by HGnFs suppresses energy

compared with that in the control group, ultimately

metabolism by the glycolytic system and is similar to aleading to the suppression of wound healing. Of note, a

low glucose environment.

small amount of ATP was produced in the group without glucose.
Energy metabolism via glycolysis in HGnFs isimportant for the wound healing of periodontal tissues.

Conflicts of Interest
The authors declare no conflicts of interest associatedwith this manuscript.

Hard tissue regeneration of alveolar bone cannot occur
without the connective tissue wound healing process

Acknowledgments

induced by HGnFs. In this respect, the intracellular andThis study was partially supported by Grants-in-Aids for
Scientific Research from the Japan Society for the Promotion of Science（21K09946, 22K17084, 22K09993 and23K19761）.

extracellular glucose metabolic environments of HGnFs
are essential.
It is important to examine the cellular energy metabolism status of HGnFs by incorporating 2-DG into theircells, as in this study, to examine wound healing in connective tissues. In future research, we would like to use
animal experiments to examine any differences

References
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Dec, 2023

129

Original Article

ODEP 3（1）：129～136，2023

Development and Clinical Evaluation of Anti-aspiration Gauze Roll
Assessment of Water Absorbency and Usability of Gauze Roll―An Exploratory Study
Chiharu KAWAMOTO, Ryotaro YAGO, Mai FUKUYAMA, Toru TANAKA＊,
Hidehiko SANO＊ and Atsushi TOMOKIYO＊
Department of Restorative Dentistry, Division of Oral Health Science, Hokkaido University Graduate School of Dental Medicine＊

Department of Restorative Dentistry, Division of Oral Health Science, Hokkaido University Faculty of Dental Medicine

Abstract
Purpose: Cotton roll is widely used in dentistry for moisture control and isolation. However, cases of airway obstruction due to aspiration of the cotton roll have been reported, and some cases have even resulted
in death. In this study, we developed an anti-aspiration gauze roll（AAGR）by attaching a thread to the gauzeroll as an alternative to conventional cotton roll to prevent aspiration and forgetting to remove the cotton
roll.
Methods: A water absorption test was conducted to evaluate the anti-moisture ability. Three types of pro, medium（250×30 mm）, and small（150×30 mm）, were prepared according tototypes, large（300×30 mm）
the size of gauze used. The length of the cotton yarn was 300 mm. An in vitro water absorption test was
conducted in accordance with the test method stipulated in the standards for medical gauze and medical cotton established by the Japanese Ministry of Health, Labour, and Welfare to evaluate the moisture control
ability of AAGR. Each specimen（n＝10）was immersed in 25℃ distilled water for 3 minutes, left on a net for
1 minute, and weighed. For statistical analysis, one-way ANOVA was performed using statistical analysis
software, followed by multiple comparison tests using the Tukey HSD test. The significance level was set at
5％. A clinical evaluation was conducted by surveying dentists about the usability of AAGR in clinical use. In
the clinical evaluation, dentists were asked to use AAGR instead of cotton roll in clinical situations and complete a questionnaire. AAGR（large）was used for adult patients, and AAGR（small）was used for pediatricpatients. The survey items included treatment details, water absorption capacity and speed, gauze portion
size, gauze thread length and thickness, ease of use, and expectations for preventing aspiration.
Results: In the water absorption test, there was a statistically significant difference in the amount of water
absorbed among each group: the amount in descending order was AAGR（large）, control group, AAGR
（medium）, and AAGR（small）. In the questionnaire survey, the results for adults showed that all items wererated as equal to or better than the conventional cotton roll, but the results for children showed that there
were areas for improvement regarding water absorption volume, water absorption speed, thickness, and easeof use.
Conclusion: The results of the water absorption test showed that AAGR（large）absorbs more water thancotton roll and has superior moisture control ability. The use of AAGR is expected to prevent both forgetting to remove moisture control tools, and their accidental ingestion or aspiration.
Key words: cotton roll, prevention of aspiration, water absorption test, questionnaire survey

Corresponding author: Chiharu KAWAMOTO, Department of Restorative Dentistry, Division of Oral Health Science, Hokkaido
University Graduate School of Dental Medicine, Kita 13, Nishi 7, Kita-ku, Sapporo 060‒8586, Japan
TEL: ＋81‒11‒706‒4261, FAX: ＋81‒11‒706‒4878, E-mail: chipa@den.hokudai.ac.jp
Received for Publication: September 14, 2023/Accepted for Publication: November 21, 2023
DOI: 10.11471/odep.2023‒015

ODEP Vol. 3, No. 1

130

In this study, to investigate whether AAGR can be

Introductionused in clinical practice as an alternative to conventional cotton roll, a water absorption test was con-Accidental ingestion and aspiration are potential

ducted to evaluate its moisture control ability, and acomplications that can occur during dental proce-clinical evaluation was made through a questionnaire

dures1）. Incident reports from FY 2012 to FY 2017 insurvey of operators regarding its usability and feel inthe Hokkaido University Hospital Dental Department

dental practice.

showed that accidental ingestion and aspiration were
the second-most reported incidents after soft tissue

The null hypothesis was that AAGR does not absorb
as much or more water as existing cotton rolls.

injuries（7 in FY 2012, 2 in FY 2013, 5 in FY 2014, 4 in
FY 2015, 3 in FY 2016, and 4 in FY 2017）.

Materials and Methods

The risk of accidental ingestion or aspiration isreported2‒4） to be particularly high in pediatric dental

1 ．Preparation of prototypes of AAGR

treatment. Cases of airway obstruction due to aspira-

Fabrication of prototypes of AAGR was outsourced

tion of cotton rolls are often reported5,6）. Moisture con-to Japan Medical Products Co., Ltd. The prototype wastrol and isolation are essential for treatment in den-made by rolling medical gauze（Japanese Pharmaco-

tistry, and rubber dam is known to be the first choicepoeia）into a roll and sewing it with cotton thread. A

7,8）

as a procedure for this purpose. However, this is not

30×300 mm rolled gauze was used as the large size,

always possible in all cases, and cotton rolls are used in

30×250 mm as the medium size, and 30×150 mm assuch cases9‒11）. While such accidents rarely occur, when

the small size. The length of the cotton yarn was 300

they do happen, the risk is very high. In fact, a casemm（Fig. 1）. Three prototypes were prepared for vari-

was reported12）in which a 2-year-old girl died at a den-ous patients, including infants, children, adults, thetal clinic in Saitama due to airway obstruction caused

elderly, and those requiring nursing care. The actual

by the aspiration of a cotton roll used during dental

prototype（Fig. 2）and a schematic diagram are shown

treatment. Therefore, when dentists use cotton rolls it

in Fig. 3. As a control group, conventional cotton rolls

is necessary to take precautions to prevent accidental（Dentalmen II, Hakujuji Co., Ltd., Japan）with cotton

ingestion and aspiration.

threads tied to them were prepared in the same man-

Another occasional problem in clinical practice is for-ner as the prototypes. The size and dimensions of eachgetting to remove the cotton roll from the oral cavity.

specimen are shown in Table 1.

Similar cases have been reported not only in dentistry

2 ．Water absorption test

but also in medical surgery; the Japan Council for Qual-

In vitro water absorption tests were conducted to

ity Health Care reported13）70 cases of gauze left in theconfirm the moisture control ability of the prototypes.

body between 2015 and 2018. However, both cotton rollThis experiment was conducted in accordance with theand gauze are indispensable for treatment in any fieldtest method stipulated in the standards for medicalof medical surgery.

gauze and medical cotton established by the JapaneseIn the context of these circumstances, we considered

Ministry of Health, Labour and Welfare15）. First, thethe development of a new tool to replace the conven-prototypes and cotton rolls（n＝10）were weighed using

tional cotton roll for the purpose of preventing aspira-a scale（AUX220, Shimadzu, Japan）. After immersing

tion and forgetting to remove the cotton roll. Initially, a

the sample in distilled water for 3 minutes, the sample

prototype was made with a thread attached to the cot-

was left on the wire mesh of a No. 10 sieve for 1 min-ton roll so that the cotton roll could be instantly

ute, allowed to drip water, and then weighed（Fig. 4）.

removed from the oral cavity by pulling the thread to

One-way ANOVA was performed on the water

prevent aspiration, but there was a concern that theabsorption test using statistical analysis software

attached thread was vulnerable to detachment. There-（SPSS28, IBM, Armonk, New York, USA）, and multiple

fore, the Anti-Aspiration Gauze Roll（AAGR）, in which

comparison tests were conducted using the Tukey HSD

a thread is sewn onto a rolled gauze, was devised andtest. The significance level was set at 5％.

14）patented

after prototype production.

Dec, 2023

30mm

Evaluation of Anti-aspiration Gauze Roll

131

300mm（Large）
or
250mm（Medium）
or
150mm（Small）
Fig. 1

Method of preparing the Anti-aspiration Gauze Rollabc
Schematic diagram of the Anti-aspiration
Gauze Roll（a）Short diameter,（b）Long diameter,（c）Length
Fig. 3Fig. 2 Prototype of the Anti-aspiration Gauze Rolla：AAGR（large）, b：AAGR（medium）, c：AAGR（small）

Table 1
SampleDimensions of each material

LengthLong diameter

Short diameter

Large

30 mm

12 mm

8 mm

Medium

30 mm

Small
Cotton Roll

30 mm
30 mm

10 mm
10 mm

7 mm
5 mm

Diameter 10 mm

Distilledwater

Weighing

Immersing

Left for 1 minute

Weighing

Fig. 4 Water absorption test method
Samples were weighed, immersed in distilled water for 3 minutes, left on a sieve wire mesh for 1 minute, and weighed again.

3 ．Questionnaire sur vey

from those of conventional cotton roll. Therefore, weCompared to conventional cotton roll, AAGR has aconducted a questionnaire survey of dentists regarding

different shape and material, so its ease of use andthe ease of use and moisture control ability of the

moisture control ability were expected to be different

AAGR instead of placement of cotton roll during dental

ODEP Vol. 3, No. 1

132

treatment. This study was conducted after obtaining
approval（021‒0180）from the Ethical Review Board forLife Science and Medical Research, Hokkaido Univer-an oral explanation of the main points of the study.
The age-sex distribution of the patients is shown in
Table 2.
AAGR（large）was used for adult patients, and AAGR

sity Hospital.
A total of 38 dentists affiliated with Hokkaido Uni-

（small）was used for pediatric patients.

versity Hospital, who agreed to participate in the ques-

Since cotton rolls are used in a variety of dental pro-tionnaire survey after receiving an explanation of thecedures, the procedures were not limited. Examples ofstudy, were enrolled in the study.

the clinical use of AAGR are shown in Fig. 5. Consent

The patients were those who went to or were hospi-

was obtained from the patient in the case of adults andtalized at Hokkaido University Hospital for dental treat-from the parent or guardian in the case of children.

ment and did not refuse to participate after receiving

The questionnaire survey included the following items:
treatment details, water absorbency, water absorption
speed, gauze portion size, thread length and thickness,

Table 2Age-sex distribution of the patientsMale（Number of participants）
Female（Number of participants）
Minimum age
Maximum age
Average age

11
27
3
77
43.55

a：Restorative treatmentease of use, and expectations for preventing aspiration（Fig. 6）.
The questionnaire survey was not statistically analyzed as it was an exploratory study.

b：Endodotic treatment

c：Pediatric dental treatment
Fig. 5 Examples of the Anti-Aspiration Gauze Roll used in clinical treatmentsa ：Composite resin filling for anterior teeth.
b ：Example of use for moisture control and isolation in a case in which a rubber dam
could not be used.
c ：The left photo shows a case in which the gauze was used during fluoride application. On the right is a case in which the gauze was applied with a seal attachedto the tip of the string and shown to the child as an applied method of treatment.

Dec, 2023

Evaluation of Anti-aspiration Gauze Roll

The amount of
Water absorption（g）

3.0
2.5

133
a
d

b

2.0
1.5

c

1.0
0.5
0

Large

MediumSmall

Cotton Roll

Fig. 7 Results of absorption test
Different letters indicate a significant difference（p＜0.05）.

Table 3Fig. 6

Questionnaire survey

Descriptive statistics

Specimens

Mean

Std. Deviation

N

Large
MediumSmall
Cotton Roll

2.4435
1.9472
1.19422.18090.10938
0.118410.07694
0.06690

10
10
10
10

Total

1.94140.4805340

Dependent variable：Amount of water absorption

Results
Other comments included:（ⅰ）There are situations
The results of the water absorption test are shown inwhen moisture control is desired, such as when attach-Fig. 7 and the details of the statistical analysis in Tableing a restoration or prosthesis to a mandibular molar,

3‒5. In the water absorption test, the results of one-way

but the risk of ingestion or aspiration makes it unavoid-

ANOVA and Tukey HSD test showed significant differ-able to perform the procedure without moisture con-ences in water absorption among the groups. Based on

trol. In such cases, this product can provide high-quality

these results, it is concluded that AAGR（large）cantreatment while minimizing the risk.（ⅱ）I would like toabsorb as much or more water as existing cotton rolls.

use this product in situations where the risk of acciden-

Therefore, the null hypothesis is rejected.

tal ingestion or aspiration is high, such as house call

The results of the survey are shown in Fig. 8. The

dentistry or when the patient is uncooperative.（ⅲ）Ittreatment details of the 38 cases included endodontic

can prevent forgetting to remove gauze during sur-treatment in 15 cases, prosthetic treatment in 10 cases,

gery.

restorative treatment in 5 cases, fissure sealant in 5
cases, hypersensitivity treatment in 2 cases, and infil-

Discussion

tration anesthesia in 1 case. The results for adults
showed that all items received a positive rating, but theIn this case, the cotton roll used at Hokkaido Univer-results for children showed that there were areas forsity Hospital was defined as the control. Cotton rolls

improvement regarding water absorption volume,

vary in size depending on the product. In this study,

water absorption speed, thickness, and ease of use.

from the viewpoint of whether AAGR can be used as a

Regarding thickness, AAGR was found to fit more eas-

substitute for cotton rolls, we considered that the water

ily into the oral cavity than conventional cotton roll.

absorbing capacity must be equal to or greater thanODEP Vol. 3, No. 1

134
Table 4
The amount ofSpecimens

Mean Difference

Water absorption

Large

MediumMedium0.4963

1.2493＊

Cotton Roll

0.2626＊－0.4963＊

Small
Cotton Roll

0.7530＊－0.2338＊

Large－1.2493＊＊－0.7530

Medium＊－0.26260.2338＊Large
MediumSig.

0.4265＜0.001

95％ Confidence Interval
Lower Bound

Upper Bound

0.38150.6112

1.13440.14771.36420.37740.42650.4265＜0.001＜0.0010.42650.4265＜0.001＜0.001－0.38150.86780.4265＜0.001－0.61120.63810.4265＜0.001－1.3642－1.13440.4265＜0.001－1.1016－0.87190.4265＜0.0010.42650.9867＊

Small

Std. Error

0.4265－0.9867＊

Cotton Roll
Cotton Roll

＊

Small
Large

Small

Multiple comparisons0.4265＜0.001＜0.001＜0.001－0.3486－0.8678－0.1189－0.6381－0.3774－0.1477

0.11890.3486

0.87191.1016

Dependent variable：Amount of water absorption
Based on observed means.
The error term is Mean Square（Error）＝0.009.
＊

The mean difference is significant at the 0.05 level.

Table 5Tukey HSD

be attributed to the size of AAGR（small）used for

SubsetSpesimens

N

1

Small
MediumCotton Roll
Large

10
10
10
10

1.1942Sig.

absorption speed, thickness, and ease of use. This may

Homogeneous subsets

a,b

2

3

4

amount of saliva and the AAGR（small）was insufficient2.18092.44351.000

assumed to be appropriate for children because of theirsmall mouths. However, children produce a large

1.9472

1.000

pediatric patients. Initially, the AAGR（small）was

1.000

1.000

Means for groups in homogeneous subsets are displayed.
Based on observed means.
The error term is Mean Square（Error）＝0.009.
a：Uses Harmonic Mean Sample Size＝10.000.
b：Alpha＝0.05.

in terms of water absorption and speed, which likely
affected its ease of use. In addition, the results of water
absorption tests showed that AAGR（small）absorbed
significantly less water than conventional cotton roll.
Based on these findings, it is considered that a gauzewith greater water absorption would be suitable forchildren. Regarding the opinion that the AAGR fits easily into the adult oral cavity, the reason may be thatconventional cotton roll is somewhat difficult to insert

that of cotton rolls. Therefore, three different sizes

between the tongue and teeth and might not be stablewere established to examine the sizes that would bewhen the space is small. However, the AAGR can be

acceptable for clinical use. AAGR
（large）absorbed more

freely adjusted due to its flexible form to fit the placewater than the conventional cotton roll of similar size,

where it is to be installed.

suggesting that it has a high moisture control effect andsufficient potential for clinical application.

The majority of dentists who participated in the survey answered that the length of the thread was“appro-

Most of the survey results, where the target patients

priate,”but there were also a few comments such as thewere adults, gave positive ratings to all items in thecurrent length is too short to wrap around the finger,

questionnaire. However, when comparing children andand concern that if the thread is too long, it may get

adults, negative opinions of cases involving children

tangled during the procedure. Most of the dentists

were noted regarding water absorption volume, water

answered that the thread thickness was“appropriate”

Dec, 2023

Evaluation of Anti-aspiration Gauze Roll

Fig. 8 Results of questionnaire survey
We conducted a questionnaire survey of 38 dentists regarding the ease of use and moisture control ability of the AAGR instead of placement of cotton roll during dental treatment.

135

ODEP Vol. 3, No. 1

136

or“thin,”and there was no response indicating it was“thick”. We expect the ease of use of the thread can be
improved by making it a little thicker. The question“Can the use of anti-aspiration gauze roll be expected
to prevent aspiration compared to conventional cotton
roll?”was answered in the affirmative by approximately 80％ of the dentists. Another commented,“I feelsafe because it has a string, so I don’t forget to pick itup.”
The survey results indicate that the use of AAGR
may help prevent accidental ingestion and aspiration.
However, the ease of use of the AAGR needs to be
improved in the future to make it even easier for dentists to use in clinical practice.
Thus, the AAGR may have the potential to be used
as an alternative to conventional cotton rolls without
placing a special burden on the dentist.

Conclusion
AAGR（large）absorbs more water than conventional
cotton rolls.
To the question“Can the use of anti-aspiration gauzeroll be expected to prevent aspiration compared to conventional cotton roll?”81％ of respondents answered in
the affirmative.
Conflict of Interest
The authors declare no conflict of interest related to thispaper.

References
1）Hou R, Zhou H, Hu K, Ding Y, Yang X, Xu G, Xue P,
Shan C, Jia S, Ma Y. Thorough documentation of theaccidental aspiration and ingestion of foreign objects
during dental procedure is necessary: review and analysis of 617 cases. Head Face Med 2016; 12: 23.
2）Fuangtharnthip P, Pujarern P, Pachimsawat P, Loeksomphot P, Janjarussakul P, Manopatanakul S. Accidental swallowing of dental objects during pediatric dental
care in Thailand. J Int Soc Prev Community Dent 2021;
11: 671‒677.
3）Zitzmann NU, Elsasser S, Fried R, Marinello CP. Foreign
body ingestion and aspiration. Oral Surg Oral Med Oral
Pathol Oral Radiol Endod 1999; 88: 657‒660.
4）Cossellu G, Farronato G, Carrassi A, Angiero F. Acci-

dental aspiration of foreign bodies in dental practice:clinical management and prevention. Gerodontology
2015; 32: 229‒233.
5）Hisanaga R, Hagita K, Nojima K, Katakura A, Morinaga
K, Ichinohe T, Konomi R, Takahashi T, Takano N, Inoue
T. Survey of accidental ingestion and aspiration at
Tokyo Dental College Chiba Hospital. Bull Tokyo Dent
Coll 2010; 51: 95‒101.
6）Sato C, Watanabe A, Ueda S, Ichiki T, Abe S. A case of
bronchial obstruction caused by a dental instrument（cotton roll）. J Jpn Soc Respir Endoscopy 2014; 36: 207.
（in Japanese）
7）Buttke TM. More on rubber dam use. J Am Dent Assoc
2016; 147: 316‒317.
8）Abreu-Placeres N, Yunes Fragoso P, Cruz Aponte P,
Garrido LE. Rubber Dam Isolation Survey（RDIS）for
adhesive restorative treatments. Eur J Dent Educ 2020;
24: 724‒733.
9）Hatirli H, Yasa B, Çelik EU. Clinical performance ofhigh-viscosity glass ionomer and resin composite on
minimally invasive occlusal restorations performed
without rubber-dam isolation: a two-year randomised
split-mouth study. Clin Oral Investig 2021; 25: 5493‒
5503.
10）Olegário IC, Moro BLP, Tedesco TK, Freitas RD, Pássaro AL, Garbim JR, Oliveira R, Mendes FM; CARDEC
03 collaborative group; Raggio DP. Use of rubber dam
versus cotton roll isolation on composite resin restorations’ survival in primary molars: 2-year results froma non-inferiority clinical trial. BMC Oral Health 2022; 22（1）: 440.
11）Carvalho TS, Sampaio FC, Diniz A, Bönecker M, Van
Amerongen WE. Two years survival rate of Class II
ART restorations in primary molars using two ways to
avoid saliva contamination. Int J Paediatr Dent 2010; 20:419‒425.
12）Nikkei Inc. A 2-year-old child died after choking on cotton roll in his throat during treatment at a dental clinic
in Saitama, Japan. https://www.nikkei.com/article/
DGXNZO09232810V10C10A6CC1000（cited 2023.9.11）
13）Japan Council for Quality Health Care. Cases of gauze
left in the body（14th Report）. https://www.med-safe.
jp/pdf/report_2018_2_R002.pdf（cited 2023.9.11）
14）Chiharu Watanabe, Japan Patent Kokai 2012‒139322（2012.7.26）
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Type=1&pageNo=1（cited 2023.9.11）

Dec, 2023

137

Original Article

ODEP 3（1）：137～143，2023

Three-dimensional Analysis of the Shaping Characteristics and Abilityof a Novel Ni-Ti Rotary File
Kazuma MATSUMOTO1, Haruna HIROSE1, Kota ISSHI2, Seishiro FUJIMASA1,
Shingo KANEMARU1, Tsukasa YANAGI3 and Etsuko MATSUZAKI1,4
1

Section of Operative Dentistry and Endodontology, Department of Odontology, Fukuoka Dental College
2

Central Dental Laboratory, Fukuoka Dental College Medical and Dental General Hospital3

Section of Oral Implantology, Department of Oral Rehabilitation, Fukuoka Dental College
4

Oral Medicine Research Center, Fukuoka Dental College

Abstract
Purpose: To compare the shaping characteristics and ability of the JIZAI（JZ）Ni-Ti rotary file to two otherNi-Ti files that have a similar operating environment to JZ, HyFlex CM（HCM）and ProTaper Next（PTN）.
Methods: Root canal preparation was performed in a J-shaped canal model using the three file systems（n＝4）, and dental cone-beam computed tomography（CBCT）was performed. The three-dimensional images
were superimposed before and after instrumentation at the same position. The X-, Y-, and Z-axes were set 1
mm coronal to the root apex, and the cross-sectional morphology, the extent of transportation, and the volume of the shaped root canal wall were evaluated.
Results: The cross-sectional morphology of the root canal was round, similar to the original root canal morphology with JZ and PTN. The HCM showed an elliptical tendency toward the sagittal direction. The transportation was relatively larger with JZ and smaller with HCM, with no significant difference between thefiles. The shaped root canal wall volume was greatest with HCM and least with JZ, with no significant difference between the files.
Conclusion: JIZAI had equivalent shaping ability to HyFlex CM and ProTaper Next in terms of transportation and shaped root canal wall volume, and therefore might be useful for safe preparation of root canals.
Key words: Ni-Ti rotary files, root canal preparation, three-dimensional imaging

Corresponding author: Etsuko MATSUZAKI, Section of Operative Dentistry and Endodontology, Department of Odontology,
Fukuoka Dental College, 2‒15‒1, Tamura, Sawara-ku, Fukuoka 814‒0193, Japan.
TEL: ＋81‒92‒801‒0411, FAX: ＋81‒92‒871‒9494, E-mail: matsuzaki@fdcnet.ac.jp
Received for Publication: September 15, 2023/Accepted for Publication: November 21, 2023
DOI: 10.11471/odep.2023‒016

ODEP Vol. 3, No. 1

138

ity and ability to follow the root canal path. Shaping

Introduction

ability is two-dimensionally（2D）evaluated by superimposing radiographs obtained before and after root canal

Ni-Ti alloy files（Ni-Ti files）are commonly used topreparation2‒5）. On the other hand, Peters et al.18）evalu-

prepare curved root canals1）. Compared to conventional

ated the surface area of the root canal by three-dimen-

stainless-steel files, Ni-Ti files have greater flexibility

sional（3D）analysis, and observed that the Ni-Ti file didand closely follow the path of the root canal due to a

not contact ≥35％ of the root canal surface area.

lower modulus of elasticity. The root canal is often

Therefore, 2D evaluation is not sufficient to explore thecurved in the apical third of the root. A stainless-steel

true shaping abilities before and after root canal prepa-

file of size #35 or more does not have sufficient flexibil-

ration. In this study, we investigated the shaping char-ity, and therefore tends to deviate from the originalacteristics and ability of JZ through a 3D analysis, andcanal pathway, resulting in transportation of the canalcompared the findings with those of HCM and PTN,

center and the formation of a ledge or zip. Convention-

which are similar to JZ in terms of the mode of rotary

ally, transportation towards the outer side and innermotion, number of files used, point size, and taper.

side of the curvature has been used to evaluate theshaping ability of Ni-Ti files2‒5）.

Materials and Methods

The low elastic modulus and breaking torque of Ni-Ti
files lead to instrument separation. Therefore, endodon-

1 ．Ni-Ti files and models

tic files should have flexibility and high fracture resis-

We used the following three types of Ni-Ti files（21tance. This has been achieved through heat treatmentmm）: JZ, #25/0.04 and #25/0.06; PTN, #17/0.04‒0.075

of the austenite phase of conventional Ni-Ti alloys6,7）.

and #25/0.06‒0.07; and HCM, #15/0.04 and #25/0.06.

One such file is HyFlex CM（HCM; Coltene, Langenau,

J-shaped epoxy resin transparent root canal models

Germany）. This file comprises the R and martensite（Dentsply Sirona, York, PA, USA; curvature, 30°; apical

phases resulting from heat treatment. When inserted

foramen diameter, #10; root canal taper, 0.02; root canalinto a curved root canal, the file does not return to its

length, 17.0 mm; n＝4）were used for root canal prepa-

original shape due to its super elasticity, and no load isration. A root canal model which did not undergo any

applied to the file8,9）. In 2012, the ProTaper Next（PTN;

instrumentation was used as the control.

Dentsply Maillefer, Ballaigues, Switzerland）file was

2 ．Root canal preparation

introduced; the offset design of the file prevents it fromThe operator was a dentist with 5 years of experi-

biting into the dentin, thereby preventing separation10）.

ence using Ni-Ti files. The working length of the root

The file is fabricated using M-wire, which is martensitic

canal was determined using a #10 stainless steel K

at room or body temperature, austenitic at 43‒50℃,（SSK; Mani）file. After setting the working length to 17

and returns to the martensitic phase when cooled,

mm and preparing a glide path with #15 and #20 SSK,

resulting in flexibility11‒13）. Recently, a file with eventhe root canal was prepared using two types of files ingreater flexibility has been manufactured through elec-

each group. An endodontic motor, Tri Auto ZX 2（J.

5,14）

. A new Ni-Ti file, JIZAI

Morita Corp., Tokyo, Japan）, was used with the auto-re-（J Z ; M a n i , T o c h i g i , J a p a n）
, which was recently

verse setting. Root canal preparation was performed bylaunched in Japan, comprises the R and/or martensite

inserting the file in the root canal three times with lightphase at room or body temperature and is used in apressure with continuous rotation of the file such thattrical discharge machining

15）

unidirectional rotation mode . Single-length prepara-

the root length shaped per insertion did not exceed 3tion was performed using two files: #25/0.04 and #25/

mm. The rotation speed and torque used were 300 rpm

0.06. This file alleviates one of the drawbacks of Ni-Ti

and 3 N（JZ）, 300 rpm and 2 N（PTN）, and 500 rpm andfiles, namely the screw-in-effect due to the cross-sec-

2.4 N（HCM）, respectively, according to the manufac-

tional shape with a land like an irregular rectangle withturer’s recommendations. The root canal was irrigateda circular arc on one side16,17）.

with distilled water using a syringe. Recapitulation with

To date, a wide range of Ni-Ti files have been intro-

a #10 SSK was performed to avoid clogging of the root

duced, and research has chiefly focused on their flexibil-

apex with debris. This cycle was repeated until the sec-Dec, 2023

3D Shaping Analysis of Three Ni-Ti Rotary FilesA

C

B
Z-axisY-axisD

139

Y-axisE

X-axisFig. 1 Three-dimensional analysis of the models after root canal preparation
Covers with three spherical reference points were placed on the models, and dental CBCT was performed（A）. The Z-axis（axial）was set to pass through the centerof the root canal and apex 1 mm coronal to the apex and the corresponding root
cross-section was considered the reference plane（B, C）. The Y-axis（sagittal）was
set along the direction of internal and external curvature perpendicular to the Z-axis（B, D）, and the X-axis（coronal）was set perpendicular to the Y-axis（D）. The intersection of the X-, Y-, and Z-axes was considered the shaping center（after instrumentation）, and the canal center（before instrumentation）（E）.

ond file reached the working length.
3 ．Dental cone-beam computer tomography（CBCT）and 3D analysis

Japan）and displayed as images using Geomagic
Freeform（3D Systems Inc., Rock Hill, SC, USA）
（Fig.
1）. The reference plane for evaluation was set 1 mm

Dental CBCT（3D Accuitomo F17, J. Morita Corp.）

coronal to the apex, and the Z-axis（axial）was set as

was performed to obtain 3D data before and after rootthat passing through the center of the root canal andcanal preparation. A radiopaque material, Calcipex Ⅱ

apex（Fig. 1B, C）. In addition, the Y-axis（sagittal）was（Nippon Shika Yakuhin Co., Ltd., Yamaguchi, Japan）,

set as the direction of internal and external curvature

was placed in the root canal using a syringe and acces-

perpendicular to the Z-axis, and the X-axis（coronal）
was

sory tip provided by the manufacturers. To preventset perpendicular to the Y-axis（Fig. 1B, D）. The inter-dead space in the model, the extrusion of Calcipex Ⅱ

section of the X-, Y-, and Z-axes was set as the shaping

from the apex was confirmed, and the syringe with thecenter, and the cross-section was considered the refer-accessory tip was tightly inserted into the root canalence plane（Fig. 1E）. The top part of Fig. 1E represents

orifice. The imaging conditions for dental CBCT were

the inner curvature, while its bottom part represents

as follows: voxel size, 125μm; 50 kW; 1.0 mA. A cover

the outer curvature. The distance of canal center trans-with three spherical reference points（Fig. 1A）was

portation and the volume of the shaped root canal wall

attached to each model prior to dental CBCT imaging.

were calculated after superimposing the 3D data before

These covers were made from a room-temperature

and after root canal preparation using Geomagic

polymerizing resin（Fixpeed, GC Corp., Tokyo, Japan）

Freeform（3D Systems Inc.）. In addition, the angle ofand Scanning Resin（Yamahachi Dental Mfg. Co., Aichi,

canal center transportation was measured by setting

Japan）, a radiopaque instant polymerizing resin. The

the internal curvature direction from the X-axis to 0‒

3D data were reconstructed using ZedView Ver. 9.0 3D

180°and the external curvature direction to 180‒360°.

planning software（ZedView, LEXI Co. Ltd., Tokyo,

ODEP Vol. 3, No. 1

140
A

C

B

D

E

90°

0°

180°

180°

0°

270°
Sample

F

90°

90°

0°

180°

270°
Angle

Sample270°
Angle

Sample

Angle

18

113

88

274

241

193

111

86

281

294

83

294

Fig. 2 Three-dimensional analysis of root canal formation with three types of files（n＝4）
Shaping cross-section with JZ（A）, HCM（B）, and PTN（C）. The black line indicates theoriginal root canal before instrumentation, and the blue line indicates the shaped root canalwall after instrumentation（A‒C）. The canal center transportation with JZ（D）, HCM（E）,
and PTN（F）. The angle of canal center transportation was measured by setting the internal curvature direction from the X-axis to 0‒180° and the external curvature direction to180‒360°, as shown at the bottom of each figure.
JZ, JIZAI Ni-Ti rotary file；HCM, HyFlex CM Ni-Ti rotary file；PTN, ProTaper Next
Ni-Ti rotary file

4 ．Statistics

similar to that in the control model. It tended to be

Median values of transportation of the canal center

elliptical after preparation with HCM, with the major

and volume of the shaped root canal wall for eachaxis along the Y-axis（Fig. 2B）.

group were analyzed by the Kruskal-Wallis test（Bell-

Next, we investigated the 3D characteristics and abil-

Curve for Excel, Social Survey Research Information

ity of the canal center transportation based on the fileCo., Ltd., Tokyo, Japan）to identify significant differ-used. Although none of the files showed a tendency forences. A p-value of ＜0.05 was considered to be statisti-

deviation in a specific direction as shown in Fig. 2D, E,

cally significant.

and F, the canal center transportation along the X-axis
was the least with PTN（Fig. 2F）. There was no char-

Results

acteristic transportation of the canal center along the
Y-axis. As shown in Fig. 3A, the median value of trans-

We examined the cross-sectional morphology of theportation of the canal center was the largest with JZ

root canal following preparation with JZ, HCM, andand the least with HCM; however, no significant differ-

PTN as an evaluation of the 3D shaping characteristics.

ences were observed between the files.

The cross-section of the root canal tended to be circularFurther, we examined the volume of the shaped root

following preparation with JZ and PTN（Fig. 2A, C）,

canal wall as an evaluation of the shaping ability. As

Dec, 2023

3D Shaping Analysis of Three Ni-Ti Rotary Files
B
0.06

0.5

0.05

Volume (mm3)Transportation of canal centersafter instrumentation (mm)

A

141

0.04
0.03
0.02
0.01
0

JZ

HCM

PTN

0.4
0.3
0.2
0.1
0

JZ

HCM

PTN

Three-dimensional analysis of the canal center transportation and the volumeof the shaped root canal wall at 1 mm coronal to the apex（n＝4）

Fig. 3The distance of canal center transportation（A）and the volume of the shaped root
canal wall（B）.
JZ, JIZAI Ni-Ti rotary file；HCM, HyFlex CM Ni-Ti rotary file；PTN, ProTaper
Next Ni-Ti rotary file

shown in Fig. 3B, the median value of volume was theportation and the Y-axis transportation. We found thatlargest with HCM and the least with JZ, with no signifi-

the Y-axis transportation was similar between the twocant difference between the files.

files, whereas the X-axis transportation tended to belarger with JZ than with PTN.

Discussion

On the other hand, HCM showed the least averagetransportation of the canal center but the largest

In this study, we analyzed the shaping characteris-amount of root canal wall removal. Compared to thetics and ability of three types of Ni-Ti files, including aother two files, the cross-section of HCM is slightly

new file. It has been observed that approximately

elliptical, with the long axis in the Y-axis direction of≥35％ of the root canal surface area does not contact

the inner and outer curvature. A previous micro-com-

the Ni-Ti file during preparation18）. This fact prompted

puted tomography study on the extent of transporta-

us to think of using 3D analysis to evaluate the shaping

tion in the direction of the internal and external curva-

characteristics and ability. Therefore, we performed 3D

ture found smaller deviation with HCM than withanalysis using highly accurate CBCT images.

PTN19）, which is consistent with the present results.

19）

A previous 3D analysis

showed that the extent of

The cross-section of the blade of HCM is triangular,

sagittal deviation was greatest at 1 mm coronal to thethat of PTN is rectangular, and that of JZ is irregularly

apex. Therefore, in this study, we analyzed the canalrectangular. PTN and JZ have an offset design in whichcenter transportation, the cross-sectional morphology,

the rotation axis is away from the center of theirand the volume of the shaped root canal wall 1 mm cor-cross-section10,16,17）. On rotation, this creates a wave-likeonal to the root apex. There was no significant differ-motion that enables shaping of the root canal wall inence in the median value of transportation of the canal

three dimensions. HCM is not a land type, and the rake

center between the files, but JZ was the largest among

angle of the file is large8,9）. The present results do not

all files. However, a 2D evaluation by Nakatsukasa etclarify whether there is a relationship between theal.15） at 1 mm coronal to the root apex showed that the

movement of the file during shaping and the morphol-

extent of deviation with JZ was slightly smaller but notogy of the shaping surface. However, in clinical settings,

significantly different from that with PTN. This dis-the canals should be carefully shaped when there is a

crepancy might be explained by the fact that the evalu-

risk of perforating the side of the inner curvature,ation equivalent to the Y-axis（sagittal）transportation

which is considered a danger zone. Therefore, clarifying

in this study was carried out by 2D analysis. In our 3D

the shaping characteristics of the file through a 3D

analysis, we evaluated both the X-axis（coronal）trans-

analysis can help prevent endodontic procedural acci-

ODEP Vol. 3, No. 1

142

dents. Regarding the morphology of the shaping

Acknowledgments

cross-section, we believe that the ideal shape is that ofThis research was partially supported by JSPS Grants-inAid for Scientific Research（JP20K9969, 23K09193）. The

the control model, which resembles the original root
canal morphology. JZ and PTN showed similar X- andY-axes deviations and shaping cross-sections as those of
the original root canal. However, relatively less shaping
along the X-axis was observed with HCM, whichshowed a large deviation along the Y-axis. In the caseauthors thank Dr. Michito Maruta（Section of Bioengineering, Department of Dental Engineering, Fukuoka Dental
College）for his assistance with the preliminary experiment.
We also thank Editage（www.editage.jp）for their English
language editing services.

of such a shape, for example, in cases requiring endodontic retreatment, residual infected dentin may be

Conflict of Interest

present due to inadequate canal preparation during the

The authors declare no conflict of interest related to thismanuscript.

initial treatment. Therefore, selecting the Ni-Ti file
based on each case may be necessary. Specifically, inbuccolingually compressed root canals, selecting JZ or
PTN may be helpful for adequate canal preparation.
The manufacturer recommends using a brushingmotion for PTN and a light pecking motion for JZ and
HCM. In this study, all three groups were operated
with a light pecking motion, and the shaping characteristics were analyzed under conditions that excluded thepossibility of the difference in motion affecting theresults. Therefore, if the PTN is operated using the
motion recommended by the manufacturer, it is possible that the results of this experiment may not match.
The shaping characteristics of Ni-Ti files have been
evaluated with dental CBCT and micro-computed
tomography with high resolution20,21）. Placement of reference points is necessary for 3D superposition; however, no well-defined method has been reported yet. In
this study, we fabricated a cover to standardize thespheres made of radiopaque Scanning Resin that serveas three reference points, and used it during CBCT
imaging.

Conclusions
In this study, we examined the 3D extent of canal
center transportation and the morphology of the shaping cross-section, and the volume of shaped root canalwall following preparation with JZ, a Ni-Ti file recently
introduced in Japan. The results of 3D analysis revealed
that JZ had equivalent shaping ability to HCM and
PTN. Therefore, JZ might be useful for safe preparation of root canals.

References
1）Zupanc J, Vahdat-Pajouh N, Schäfer E. New thermomechanically treated NiTi alloys―a review. Int Endod J
2018; 51: 1088‒1103.
2）Lu Shi, Wagle S. Comparing the centering ability of different pathfinding systems and their effect on final
instrumentation by Hyflex CM. J Endod 2017; 43: 1868‒
1871.
3）Silva EJ, Tameirão MD, Belladonna FG, Neves AA,
Souza EM, De-Deus G. Quantitative transportationassessment in simulated curved canals prepared withan adaptive movement system. J Endod 2015; 41: 1125‒
1129.
4）Saleh AM, Vakili Gilani P, Tavanafar S, Schäfar E. Shaping ability of 4 different single-file systems in simulated
S-shaped canals. J Endod 2015; 41: 548‒552.
5）Özyürek T, Yılmaz K, Uslu G. Shaping ability of Reciproc, WaveOne GOLD, and HyFlex EDM single-file systems in simulated S-shaped canals. J Endod 2017; 43:805‒809.
6）Kim HC, Yum J, Hur B, Cheung GS. Cyclic fatigue andfracture characteristics of ground and twisted nickel-titanium rotary files. J Endod 2010; 36: 147‒152.
7）Zhou HM, Shen Y, Zheng W, Li L, Zheng YF, Haapasalo
M. Mechanical properties of controlled memory andsuperelastic nickel-titanium wires used in the manufacture of rotary endodontic instruments. J Endod 2012; 38:1535‒1540.
8）Santos Lde A, Bahia MG, de Las Casas EB, Buono VT.
Comparison of the mechanical behavior between controlled memory and sperrelastic nickel-titanium files via
finite element analysis. J Endod 2013; 39: 1444‒1447.
9）Gündoğar M, Özyürek T. Cyclic fatigue resistance ofOneshape, Hyflex EDM, Wave One Gold, and Resiproc
Blue nickel-titanium instruments. J Endod 2017; 43:1192‒1196.

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3D Shaping Analysis of Three Ni-Ti Rotary Files

14310）Mittal R, Singla MG, Garg A, Dhawan A. A comparisonof rotary triple helix root canal instruments. J Endodof apical bacterial extrusion in manual, ProTaper rotary,
and One Shape rotary instrumentation techniques. J17）Ha JH, Park SS. Influence of glide path on the screw-in

Endod 2015; 41: 2040‒2044.
11）Alapati SB, Brantley WA, Iijima M, Clark WA, Kovarik
L, Buie C, Johnson WB. Metallurgical characterizationof a new nickel titanium wire for rotary endodontic
instruments. J Endod 2009; 35: 1589‒1593.
12）Ye J, Gao Y. Metallurgical characterization of M-Wirenickel titanium shape memory alloy used for endodontic
rotary instruments during low-cycle fatigue. J Endod
2012; 38: 105‒107.
13）Peteira ES, Gomes RO, Leroy AM, Singh R, Peters OA,
Bahia MG, Bahia MG. Mechanical behavior of M-Wireand conventional NiTi wire used to manufacture rotary
endodontic instruments. Dent Mater 2013; 29: e318‒324.
14）Gomaa MA, Osama M, Badr AE. Shaping ability of threethermally treated nickel-titanium systems in S-shaped
canals. Aust Endod J 2021; 47: 435‒441.
15）Nakatsukasa T, Ebihara A, Kimura S, Maki K, Nishijo M,
Tokita D, Okiji T. Comparative evaluation of mechanicalproperties and shaping performance of heat-treatednickel titanium rotary instruments used in the single-length technique. Dent Mater J 2021; 40: 743‒749.
16）Diemer F, Calas P. Effect of pitch length on the behavior

2004; 30: 716‒718.
effect and torque of nickel-titanium rotary files in stimulated resin root canals. Restor Dent Endod 2012; 37:215‒219.
18）Peters OA, Schönenberger K, Laib A. Effects of four
Ni-Ti preparation techniques on root canal geometry
assessed by micro computed tomography. Int Endod J
2001; 34: 221‒230.
19）Huang Z, Quan J, Liu J, Zhang W, Zhang X, Hu X. A
microcomputed tomography evaluation of the shaping
ability of three thermally-treated nickel-titanium rotary
file systems in curved canals. J Int Med Res 2019; 47:
325‒334.
20）Pasqualini D, Alovisi M, Cemenasco A, Mancini L,
Paolino DS, Bianchi CC, Roggia A, Scotti N, Berutti E.
Micro-computed tomography evaluation of ProTaper
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21）Gagliardi J, Versiani MA, de Sousa-Neto MD, Plazas-Garzon A, Basrani B. Evaluation of the shaping characteristics of ProTaper Gold, ProTaper NEXT, and ProTaper
Universal in curved canals. J Endod 2015; 41: 1718‒1724.

ODEP Vol. 3, No. 1

144

Original Article

ODEP 3（1）：144～151，2023

Inhibition of Intracellular Glucose Metabolism
by 2-deoxyglucose Suppresses Proliferation and Differentiationof Mouse Osteoblast-like Cells
Hirohito KATO, Yoichiro TAGUCHI, Runbo LI, Chiaki MANDAI, Masahiro NOGUCHI, Kei SHIOMI,
Hideki AKIMOTO, Sho MIZUTANI, Tomoko KANDA, Chizuko OGATA and Makoto UMEDA
Department of Periodontology, Osaka Dental University

Abstract
Purpose: Glucose metabolism plays a critical role in osteoblast differentiation and influences vital processes
involved in bone formation and remodeling. This study explored the inhibitory effects of 2-deoxyglucose（2-DG）on glucose uptake by osteoblasts and its impact on cell differentiation.
Methods: Based on the structural similarity between 2-DG and glucose, the effects of glucose metabolism
on osteoblast differentiation were examined by competitive inhibition of glucose uptake. MC3T3-E1（mouse
osteoblast-like cells）were cultured in media containing 10％ FBS supplemented with 2-DG to evaluate cellproliferation and osteogenic differentiation.
Results: Inhibition of intracellular glucose metabolism by 2-DG inhibited MC3T3-E1 cell proliferation andmineralization. However, the gene expression of Runx2 mRNA was enhanced in the 2-DG group. Moreover,there was a negligible difference in the osteocalcin production.
Conclusion: Decreased intracellular glucose metabolism inhibited osteoblast proliferation and differentiation.
Key words: glucose metabolism, osteoblasts, differentiation

Corresponding author: Yoichiro TAGUCHI, Department of Periodontology, Osaka Dental University, 8‒1, Kuzuha-hanazono-cho, Hirakata, Osaka 573‒1121, Japan
TEL: ＋81‒72‒864‒3084, FAX: ＋81‒72‒864‒3184, E-mail: taguchi@cc.osaka-dent.ac.jp
Received for Publication: September 15, 2023/Accepted for Publication: November 27, 2023
DOI: 10.11471/odep.2023‒017

Dec, 2023

2-deoxyglucose Affects the of Mouse Osteoblast-like Cells

145

culture studies. MC3T3-E1 cells were purchased from

Introduction

the Cell Engineering Division of RIKEN BioResource

Bones play a crucial physiological role in supporting

Eagle’s Medium（MEM; Nacalai Tesque, Kyoto, Japan）,

bodily movements, storing essential minerals such as

supplemented with 10％ fetal bovine serum（FBS;

calcium and phosphorus, and functioning as hematopoi-

HyClone, Thermo Fisher Scientific, MA, USA）and anti-

Research Center（Tsukuba, Japan）, cultured in Modified

1）

etic organs . Bone tissue constantly undergoes remod-

biotics（100 U/mL penicillin, 100μg/mL streptomycin,

eling, with a continuous interplay between osteoblasts

and 25μg/mL amphotericin B; Nacalai Tesque）at 37℃

responsible for bone formation and osteoclasts responsi-

under 5％ CO 2 . The culture medium was changed

ble for bone resorption, ensuring the structural integ-

every three days, and when the cells reached sub-con-rity and strength of the bones

2‒5）. Therefore, it isfluence, they were harvested and subcultured. Cells

important to elucidate the mechanisms regulating bone

were used between passages 3 and 6 for each experi-

metabolism in periodontology. Alveolar bone regenera-

ment, according to our previous research methods14）.

tion is crucial in the success or failure of periodontal
tissue regeneration therapy in periodontal treatment.

2 ．Conditions for culture medium with 2-DG
solution

Recent research has suggested that glucose metabolism

MC3T3-E1 cells were cultured in MEM supple-

plays a significant role in osteoblast differentiation6‒8）.

mented with 10％ FBS and a medium with a glucose

Glucose serves as the primary substrate for cellularconcentration of 100 mg/dL was prepared with fourenergy production and influences various cellular func-

2-DG concentrations（0.05, 0.1, and 0.2 mM）dissolved intions. Glucose uptake by osteoblasts has emerged as a

the medium. The concentration of 2-DG was selected by

critical regulatory checkpoint for osteoblast maturation

setting the highest concentration（0.2 mM）and diluting

and differentiation

9,10）. Therefore, glucose metabolism

it two-fold with reference to the results of the cell pro-

may also be important in bone resorption in periodonti-

liferation. In this study, medium with a glucose concen-

tis and bone regeneration in periodontal tissue regener-

tration of 100 mg/dL was used as a positive control andation therapy.

medium with a glucose concentration of 0 mg/dL as a2-Deoxyglucose（2-DG）shares structural similarities

negative control. Osteogenic differentiation assays werewith glucose but cannot be metabolized intracellularly,

performed using osteogenic medium containing 50μM

competitively inhibiting glucose uptake. The character-

L-ascorbic acid 2-phosphate（Nacalai Tesque）, 10 mM

istics of 2-DG are being researched for various applica-

β-glycerophosphate（Wako Pure Chemical Industries,

tions, such as cancer therapy and immunotherapy11‒13）.

Tokyo, Japan）, and 10 nM dexamethasone（Wako Pure

Therefore, it is important to investigate the effects ofChemical Industries）as previously described15）.

inhibiting glucose metabolism using 2-DG on osteoblast
differentiation.

3 ．Cell proliferation
The MC3T3-E1 cells were plated and adhered forThis study elucidated the importance of glucose

24 h. The medium was replaced with medium with ormetabolism in osteoblasts using osteoblast lineage cells,

without 2-DG, and the cells were incubated for 24, 72,

and the effect of 2-DG on osteoblast differentiation wasand 120 h. Cell proliferation was determined using Cellclarified by inhibiting glucose uptake. The study may

Count Reagent SF（Nacalai Tesque）by measuring thethus contribute to a better understanding of the regula-amount of formazan. The absorbance was measured attory mechanisms governing bone formation and metab-

450 nm and the data were analyzed using SoftMax Pro

olism by providing essential insights into glucose

software（Molecular Devices, CA, USA）.

metabolism in osteoblasts.

4 ．Live cell staining
MC3T3-E1 cells were cultured in 24-well plates at aMaterials and Methods

density of 2×103 cells/mL for 24 h. Subsequently, themedium was changed to a medium with or without

1 ．Cell culture

2-DG for 24, 72, and 120 h. At each time point, live cells

MC3T3-E1 cells are immortalized mouse osteo-

were stained for 15 min at 37℃ with 4μM Calcein-AM

blast-like cells that are commonly used for bone-related

solution（Dojindo Molecular Technologies Inc., MD,ODEP Vol. 3, No. 1

146

USA）
. Calcein-stained images were acquired using an

hyde phosphate dehydrogenase.

all-in-one fluorescence microscope（BZ-9000, Keyence,

MC3T3-E1 cells were cultured in osteogenic DMEM,

Osaka, Japan）, and the percentage of positively stainedwith or without 2-DG, for 3 weeks. The culture super-areas in the images was determined using ImageJ soft-

natant was collected and the quantity of OCN wasware（National Institutes of Health, MD, USA）.

quantified using an OCN detection kit（TAKARA Bio

5 ．Cell migration assay
In this study, cell migration assay was performed

Inc.）according to the manufacturer’s instructions.
9 ．Statistical analysis

using a wound repair assay kit（Ibidi GmbH, Martin-

Data are presented as the means±SD. One-way anal-

sried, Germany）. MC3T3-E1 cells were seeded at 3.5×

ysis of variance followed by Tukey’s post hoc test was

104 cells/70μL in a cell culture insert. After the cells

used to determine significance. p values＜0.05 werereached confluence, the culture insert was removed andconsidered significant. Statistical analyses were per-the medium was replaced with a serum-free medium

formed using IBM SPSS Statistics Ver. 17（IBM, IL,

containing 2-DG. Images of each wound were taken atUSA）.

0, 12, and 24 h using a BZ-II all-in-one fluorescencemicroscope（Keyence）. ImageJ software（National Insti-

Results

tutes of Health）was used to measure the denuded area
in each image. Data are presented as the percentage ofhealed wound area at 12 and 24 h compared with theinitial wound at 0 h.

1 ．Cell proliferation
The number of live cells stained with Calcein also
decreased in the 2-DG group（Fig. 1A）. According to the

6 ．Alkaline phosphatase（ALP）staining

formazan test in the cell proliferation assay, the 2-DG

MC3T3-E1 cells were seeded in osteogenic Dulbecco’s

group significantly suppressed the proliferative ability

MEM（DMEM）with or without 2-DG and cultured forof MC3T3-E1 cells（Fig. 1B）.

1 or 2 weeks. To assess ALP activity, ALP staining was

2 ．Cell migration

performed at each time point using an ALP staining kit

Cell migration was examined using a scratch assay;（Sigma-Aldrich, MO, USA）
. In addition, staining inten-

cell migration to the wound was significantly sup-sity of ALP staining was evaluated using ImageJ soft-pressed in the 2-DG（0.2 mM）-treated group comparedware（National Institutes of Health）.

to that in the control group（Fig. 2A）. The migration

7 ．Alizarin Red S（ARS）staining

ability showed a significant tendency to be gradually

MC3T3-E1 cells were cultured in osteogenic DMEM

suppressed over time, and a concentration-dependent

with or without 2-DG for 2 or 3 weeks. To assess the

suppression tendency was observed in the 2-DG-added

mineralization, the cells were stained with 1％ ARS

group after 24 h（Fig. 2B）. There was a statisticallysolution（Wako Pure Chemical Industries）for 3 min atsignificant difference in closure rates between the 0.05

room temperature and washed three times with phos-mM test group and the other test groups, but no signif-

phate-buffered saline. In addition, staining intensity of

icant difference from the positive control of 100 mg/dL

ARS staining was evaluated using ImageJ software

was identified.

（National Institutes of Health）.
8 ．Runx2 mRNA gene expression and osteocalcin（OCN）measurement
Whole-cell RNA was isolated using the RNeasy Mini

3 ．ALP activity and mineralization
ALP staining intensity and activity were suppressedin the 2-DG-added group（Fig. 3）. The number of calcifications stained with ARS was lower in the 2-DG-treated

Kit（Qiagen, Venlo, the Netherlands）and PrimeScript

group（Fig. 4）. ALP staining intensity at weeks 1 and 2

RT Reagent Kit（TAKARA Bio Inc., Kusatsu, Japan）

of incubation and ARS staining intensity at week 3 ofaccording to the manufacturer’s instructions. Gene

incubation were lower than the negative control. This

expression was examined by real-time PCR assays

could be due to the strong inhibition of glucose metabo-（Thermo Fisher Scientific）. mRNA expression oflism by 2-DG.

Runx2 was evaluated using quantitative real-time PCR
in accordance with standard protocols. Gene expression
was normalized to the housekeeping gene glyceralde-

4 ．Runx2 mRNA gene expression and OCN production
The gene expression of Runx2 significantly increased

Dec, 2023
A
Glucose
2－DG

2-deoxyglucose Affects the of Mouse Osteoblast-like Cells
100
0

100
0.05

100
0.1

100
0.2

0
0

147

（mg/dL）
（mM）

24 h

72 h

120 h

B
Cell proliferation（Absorbance）

1.5

Glucose2－DG
100 mg/dL＋ 0 mM
100 mg/dL＋0.05 mM
100 mg/dL＋ 0.1 mM
100 mg/dL＋ 0.2 mM
0 mg/dL＋ 0 mM

1.0

0.5

A
Glucose
2－DG

24

100
0

††
††

72

100
0.05

Fig. 1 Effect of 2-DG on cell proliferation in MC3T3-E1（A）The number of viable cells（as detected through Calcein-AM staining）decreased in the 2-DG-added group and 0 mg/dL

††

††
††††

††††††
0.0†

120

group（negative control）（B）.
The OD value of Cell Count Regent
SF was assessed after culturing for 24, 72, and 120 h. Scale bars
＝500μm. †：＜0.05 versus control group. ††：＜0.01 versus con-trol group. Each experiment was performed three times for sta（h） tistical analysis（n＝3）.

100
0.1

100
0.2

0
0

（mg/dL）
（mM）

0h

12 h

24 h

Wound closure（％）

B 50
40
30

Glucose2－DG
100 mg/dL＋ 0 mM
100 mg/dL＋0.05 mM
100 mg/dL＋ 0.1 mM
100 mg/dL＋ 0.2 mM
0 mg/dL＋ 0 mM

20

††

10
0

††

††
12

††

24

Fig. 2 Effect of 2-DG on cell migration in MC3T3-E1（A）PDLCs were grown in a cell culture insert. After the cells
reached confluence, the culture insert was removed and the
medium was changed to a medium containing 2-DG. The final-toinitial wound size ratio represents the change in wound area.
（B）
The percentage of wound-healed area was measured after 0, 12,
and 24 h. ††：＜0.01 versus control group. Each experiment was（h） performed three times for statistical analysis（n＝3）.

ODEP Vol. 3, No. 1

148
A
Glucose
2－DG

100
0

100
0.05

100
0.1

100
0.2

0
0

（mg/dL）
（mM）

1w

2w

B
ALP stain intensity

30

2－DG
Glucose100 mg/dL＋ 0 mM
100 mg/dL＋0.05 mM
100 mg/dL＋ 0.1 mM
100 mg/dL＋ 0.2 mM
0 mg/dL＋ 0 mM

20

10
††

0

†

††
†† ††

††
††

1

2

††
（w）

Fig. 3 Effect of 2-DG on ALP activity in MC3T3-E1（A）The intensity of ALP staining was decreased in the 2-DG-added groups after 1 or 2 weeks.
（B）Quantitative results were evaluated by Image J. †：＜0.05 versus control group. ††：＜0.01
versus control group. Each experiment was performed three times for statistical analysis（n＝3）.

in the 2-DG（0.2 mM）group（Fig. 5A）. OCN production

m i g r a t i o n o f h u m a n g i n g i v a l f i b r o b l a s t s 1 9）. T h e

was slightly enhanced in the 2-DG-treated group; how-observed inhibitory effects on cell proliferation andever, there was only a small difference in production

migration suggest that 2-DG could potentially interfere（Fig. 5B）.

with the essential processes involved in bone formation
and repair.
ALP is a specific marker of osteoblast differentiation

Discussion

and is expressed from early to mid-differentiation20）.
2-DG was used to investigate the effects of glucose

The present study found that ALP activity was sup-metabolism on osteoblast differentiation. 2-DG is takenpressed by the addition of 2-DG. The significant reduc-up by intracellular glucose transporters. Studies havetion in ALP activity in 2-DG-treated osteoblasts further

reported that cells with a high glucose uptake capacity,

supports the conjecture that 2-DG negatively affects

such as osteoblasts and osteoclasts, are also character-

mid-stage osteoblast differentiation. This is consistent

16,17）. The present

with studies in other cell types, in which glycolysis inhi-

study showed that the 2-DG-induced impairment of glu-

bition has been associated with reduced ALP activity21）.

cose metabolism suppressed the biological functions ofThus, the inhibition of glucose metabolism may affectmouse osteoblasts.

mid-stage osteoblast differentiation by suppressing ALP

ized by a high 2-DG uptake capacity

2-DG inhibited the proliferation and migration ofmouse osteoblasts. Glucose metabolism regulates osteo18）

activity.
Mineralization has been evaluated as a marker of latesarcoma cell growth . In addition, our previous study

osteoblast differentiation. Our results showed that theshowed that glucose metabolism was involved in the

addition of 2-DG significantly suppressed mineralization.

Dec, 2023

2-deoxyglucose Affects the of Mouse Osteoblast-like Cells

A
Glucose
2－DG

100
0

100
0.05

100
0.1

149

100
0.2

0
0

（mg/dL）
（mM）

2w

3w

B

35

Glucose2－DG
100 mg/dL＋ 0 mM
100 mg/dL＋0.05 mM
100 mg/dL＋ 0.1 mM
100 mg/dL＋ 0.2 mM
0 mg/dL＋ 0 mM

ARS stain intensity

30
25
20

††

15
10

††

5
0

††

††

††
††

†† ††

2

3

（w）

Fig. 4 Effect of 2-DG on mineralization in MC3T3-E1（A）Calcified nodule formations stained with ARS were significantly decreased in the 2-DG-addedgroups.（B）Quantitative results were evaluated by Image J. ††：＜0.05 versus control group. Eachexperiment was performed three times for statistical analysis（n＝3）.
B

Runx2 mRNA expression

2.5

＊＊

OCN production（ng/mL）

A

2.0
1.5
1.0

＊
†

0.5

0.0
Glucose 100 100 100 100
2－DG 0 0.05 0.1 0.2

0 （mg/dL）
0 （mM）

30

＊

＊

20

10

0
Glucose 100 100 100 100
2－DG 0 0.05 0.1 0.2††

0 （mg/dL）
0 （mM）

Fig. 5 Effect of 2-DG on Runx2 mRNA expression and OCN production in MC3T3-E1（A）Runx2 mRNA expression in the 2-DG-added groups significantly increased or
decreased depending on the concentration of 2-DG after 1 week.（B）Osteocalcin（OCN）
production in the 2-DG-added groups increased after 3 weeks. A significant decrease compared with the control group is indicated by †：＜0.05, ††：＜0.01. A significant increase
compared with the control group is indicated by ＊：＜0.05, ＊＊：＜0.01. Each experiment

was performed three times for statistical analysis（n＝3）. The expression of Runx2 mRNAwas evaluated as the ratio to expression at 100 mg/dL glucose concentration.

ODEP Vol. 3, No. 1

150This result is similar to that of previous studies show-

potential clinical implications of these findings in rela-ing that bone formation is promoted by glucose trans-

tion to bone-related diseases and therapies.

22）

porters such as GLUT 1, 3, and 4 . Therefore, glucose
metabolism is a key factor in bone mineralization.

Conclusions

Runx2 is a marker of early differentiation in osteoblast maturation and it is also an essential transcription
factor in osteoblasts

23,24）

The findings of this study suggest that inhibitory. OCN is considered the mosteffects of 2-DG on osteoblast proliferation, migration,

recently identified differentiation marker of osteoblasts

and osteogenic differentiation markers may have poten-

among osteogenic markers

25）. The present study

tial negative effects on bone formation.

revealed unexpected results regarding Runx2 and OCN.
Although Runx2 expression and OCN production were

Conflict of interest

significantly enhanced in the 2-DG-added group, there

The authors declare no conflicts of interest associatedwith this manuscript.

was no particularly large difference in OCN production.
The unexpected upregulation of Runx2 gene expression
in the 2-DG-treated group warrants further investiga-

Acknowledgementstion. Inhibition of glucose metabolism by 2-DG may

This study was partially supported by Grants-in-Aid for
Scientific Research from the Japan Society for the Promotion of Science（C）（21K09946, 22K17084, and 22K09993）andthe Osaka Dental University Research Promotion Grant（23‒10）.

increase expression of Runx2 as a result of the low
degree of differentiation. Inhibition of early differentiation markers may indicate impaired osteoblast maturation, but 2-DG may not affect late osteoblast functionality, as there were significant but not large differencesin OCN production. This observation suggests that theeffects of 2-DG on osteoblast differentiation are selective and may vary at different stages of the differentiation process. Runx2 is a gene that is expressed in osteoblasts in the early differentiated state. In this study,
treatment with 0.2 mM 2-DG increased Runx2 mRNA
expression. We speculate that the increased expressionof Runx2 led to a low degree of differentiation and suppressed the formation of calcified nodules, which are an
indicator of late differentiation. However, a detailed
analysis using various molecular biological techniques
would be required to demonstrate this phenomenon.
We will examine the mechanisms involved in theincreased mRNA expression of Runx2 and OCN production in future research. The finding of the present
study that 2-DG inhibits osteoblast proliferation and differentiation may lead to the discovery of a therapy
through the regulation of glucose metabolism.
This study investigated the effects of 2-DG on osteoblast proliferation, migration and osteogenic differentiation. However, the role of glucose metabolism itself inosteoblasts remains to be elucidated. This study used
2-DG and osteoblasts to determine the importance of
glucose metabolism in bone formation. However, further studies are required to elucidate the molecularmechanisms underlying these effects and explore theReferences
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ODEP Vol. 3, No. 1

152

Case Report

ODEP 3（1）：152～157，2023

Risk of an Immediate-type Allergy to Minocycline Hydrochloride Ointment
Maki HOSOKI1, Yoshifumi OKAMOTO2, Toyoko TAJIMA1, Mayu MIYAGI1, Yoshiaki KUBO3,
Maiko SOGAWA3, Resmi RAJU4, Shaista AFROZ5 and Yoshizo MATSUKA1
1

Department of Stomatognathic Function and Occlusal Reconstruction,
Tokushima University Graduate School of Biomedical Sciences2

Tokushima Prefecture Dental Association

3

Department of Dermatological Science, Tokushima University Graduate School of Biomedical Sciences
4

Eunice Kennedy Shriver National Institute of Child Health and Human Development（NICHD）,
National Institutes of Health（NIH）
5

Department of Prosthodontics and Dental Material, Aligarh Muslim University（India）

Abstract
Minocycline hydrochloride ointment is applied to periodontal pockets to treat periodontal disease. We
encountered a case of an immediate-type allergy to minocycline hydrochloride dental ointment, despite theabsence of a history of allergies to antibiotics.
Case report: The patient was a 72-year-old female, with no significant medical history. Systemic erythemaand urticaria developed in June 2017. Similar symptoms occurred again in October and November 2017, forwhich she consulted for an internal medicine examination. However, the cause remained unknown. Because
the symptoms always occurred after she received treatment for periodontal disease, we performed a prick
test to diagnose any possible allergy caused by the materials used in her dental treatment. A positive reaction to the allergy test was reported for minocycline hydrochloride ointment. We informed her dentist andrequested that periodontal maintenance treatment with minocycline hydrochloride ointment be ceased.
Course: She has not exhibited any allergic symptoms since the application of minocycline hydrochloride
ointment was stopped.
Discussion: It was suspected that allergic symptoms could have developed during the course of periodontal
maintenance treatment. Given that the incidence of allergic reactions is increasing, it is important to consider
the possibility of allergic reactions to dental materials and medicaments.
Conclusion: An incremental application of a small amount of topical dental antibiotics into the periodontal
pocket can result in immediate-type allergic reaction.
Key words: allergy, urticaria, minocycline hydrochloride ointment

Corresponding author: Yoshizo MATSUKA, Department of Stomatognathic Function and Occlusal Reconstruction, Tokushima
University Graduate School of Biomedical Sciences, 3‒18‒15, Kuramoto-cho, Tokushima 770‒8504, Japan
TEL: ＋81‒88‒633‒7350, FAX: ＋81‒88‒633‒7391, E-mail: matsuka@tokushima-u.ac.jp
Received for Publication: November 21, 2022/Accepted for Publication: March 27, 2023
DOI: 10.11471/odep.2023‒018

Dec, 2023

Hypersensitivity to Dental Antibiotics

153

mandibular joint disorder in 2005. She had consulted

Introduction

several dentists for regular dental treatment over theyears.

Minocycline is a semisynthetic, second-generation tet-

In February 2017, the patient consulted a new dental

racycline derivative and is used worldwide for theclinic for the treatment of periodontal disease. She

treatment of acne. The mechanism of action is the inhi-

received general periodontal maintenance treatment

bition of bacterial protein synthesis. It does not act on（scaling, root planing and polishing）. A panoramic den-animal ribosome 80S, but specifically acts on bacterial

tal radiograph（Fig. 1）of the patient taken in February

ribosome 70S, so that the aminoacyl tRNA is inhibited2017 revealed five dental implants in the mandibularfrom binding to the mRNA ribosome complex. The anti-

molar area. No significant pathological findings werebacterial spectrum is broad, and includes gram positive

observed around the implants. Alveolar bone resorption

and negative bacteria, rickettsia, and chlamydia. The

was observed around the molars, especially around theantibacterial activity is one to four times stronger thanright lower second molar（Table 1）.

that of tetracycline. It has also been reported to be

The history of her present illness was as follows. In

effective as an adjunct to scaling and root planing1‒3）.

June 2017, she visited a dental clinic at around 16：00,

However, there are many reports of pigmentation as

where she received periodontal maintenance treatment.

a side effect of minocycline4‒11）. There are also some

She returned home around 17：00. Later that day, the12‒15）. Additionally, minocycline ispatient noticed urticaria all over her body, except forcontraindicated in patients with a history of hypersensi-

her face. Because the hospital was closed, she pur-tivity to other drugs in the tetracycline group of antibi-chased over-the-counter medicine for local application

otics such as tetracycline and doxycycline15,16）. How-and oral administration from a drugstore. Fortunately,

ever, there have been fewer reported cases of allergy

her symptoms were relieved after taking the medicine.

to minocycline than to other antibiotics such as penicil-

Similar symptoms recurred in the months of Octoberlin and cephalosporin17）.

and November 2017. At that time she consulted a phy-reports of anaphylaxis

We report a case of immediate-type allergy to mino-

sician and received an internal medicine examination.

cycline hydrochloride dental ointment 2％（Periofeel

According to the physician, erythema and urticaria

Dental Ointment 2％, Showa Yakuhin Kako Co., Tokyo,

were observed throughout her body with the concomi-

Japan）, used at the time of periodontal treatment, eventant symptoms of nausea and diarrhea. The patient wasthough the patient had no history of allergy to any anti-

treated with betamethasone sodium phosphate（RIN-biotic.

DERON Injection, Shionogi Healthcare Co., Osaka,

Consent was obtained from the patient for publica-

Japan）and metoclopramide hydrochloride（Primperantion. After obtaining consent, we inquired about treat-

Injection, Nichi-Iko Pharmaceutical Co., Ltd., Toyama,

ment details from the internist.

Japan）drips, and was prescribed prednisolone（PREDONINE Tablets, Shionogi Healthcare Co.）5 mg, Fex-

Case Reportofenadine hydrochloride（Allegra Tablets, Sanofi K. K.,Tokyo, Japan）2T, and l-cysteine（HYTHIOL Tablets,

A 72-year-old Japanese female who developed recur-

Hisamitsu Pharmaceutical Co., Inc., Saga, Japan）
1T, andrent allergic symptoms following each session of treat-

her symptoms seemed to subside. Her diagnosis wasment for periodontal disease was referred for anrecorded as idiopathic urticaria. The patient noticed

allergy test, to rule out the possibility of allergic reac-that the allergy recurred after each dental treatment

tions to any dental materials, at Tokushima University

and consulted her family dentist, who referred the

Hospital in June 2017.

patient to the dental metal allergy clinic of Tokushima

Her medical history was not significant except for

University Hospital for allergy testing.

insomnia and there was no history of allergic disease.

Allergy testing for the dental materials was planned

History of dental treatments included dental implantand progress was monitored. There were seven dental

surgery（bilateral mandibular molars）in 2003, treat-

medicaments that had been used in the dental clinic: six

ments for intra-oral candidiasis in 2005, and temporo-kinds of toothpaste and minocycline hydrochloride den-ODEP Vol. 3, No. 1

154

Fig. 1 Panoramic dental radiograph in February 2017
Alveolar bone resorption was observed around the molars, especially around theright lower second molar.

2017 February

2017 March2017 June2017 October

Mobility
BOP
EPP

Table 1

Periodontal examination

0
＋
4

0
－
4

0
－
3

0
－
3

0
－
3

0
－
3

0
－
3

0
－
3

0
－
3

0
－
3

0
＋
4

0
＋
5

0
＋
4

0
－
4

7

6

5

4

3

2

1

1

2

3

4

5

6

7

3
－
0

3
－
0

3
－
0

3
－
0

3
－
0

3
－
0

4
－
0

/

EPP
BOP
Mobility

6＋0

Mobility
BOP
EPP

0
－
4

0
＋
4

0
－
3

0
－
3

0
－
3

0
－
3

0
－
3

0
－
3

0
－
3

0
－
3

0
－
3

0
－
4

0
＋
4

0
＋
4

7

6

5

4

3

2

1

1

2

3

4

5

6

7

3
－
0

3
－
0

3
－
0

3
－
0

3
－
0

3
－
0

4
－
0

/

EPP
BOP
Mobility

6＋0

Mobility
BOP
EPP

0
＋
4

0
＋
4

0
－
3

0
－
3

0
－
3

0
－
3

0
－
3

0
－
3

0
－
3

0
－
3

0
＋
3

0
＋
3

0
＋
4

0
＋
4

7

6

5

4

3

2

1

1

2

3

4

5

6

7

3
－
0

3
－
0

3
－
1

3
－
1

3
－
0

3
－
0

3
－
0

/

EPP
BOP
Mobility

7＋
0

Mobility
BOP
EPP

0
－
3

0
－
3

0
－
3

0
－
2

0
－
3

0
－
3

1
－
3

1
－
3

0
－
3

0
－
3

0
－
2

0
－
2

0
－
3

0
－
3

7

6

5

4

3

2

1

1

2

3

4

5

6

7

3
－
0

3
－
0

2
－
1

2
－
1

2
－
0

3
－
0

3
－
0

EPP
BOP
Mobility

6－0Periodontal treatment reduced inflammation and periodontal pockets slightly.
Examination of Periodontal Pocket（EPP）, Bleeding on Probing（BOP）/

Dec, 2023

Hypersensitivity to Dental Antibiotics

155

likely to cause drug allergy. Symptoms include itching,rash, fever, asthma, anaphylaxis, and severe forms ofdrug reactions such as Stevens-Johnson syndrome（SJS）
and toxic epidermal necrolysis（TEN）. Most drug allergies are classified as type I allergies. However, some
Acidulated phosphatefluoride Jelly
Minocyclinehydrochloride ointment

drugs can also cause delayed allergic reactions, known
as type IV allergies. In this case, based on the symptoms and test results, we concluded that a type I
allergy was more likely than a type IV allergy. Diagnosis is established clinically by using a skin test. The
first step following a positive allergic reaction test is toFig. 2 The results of a prick test
The results of the prick test after 30
minutes are shown. Minocycline hydrochloride ointment showed a positive
reaction. Eventually. acidulated phosphate fluoride jelly showed a negative
reaction.

stop the use of any of the causative agents or drugs,
followed by the administration of antihistamines andsteroid drugs as needed17）.
Allergy to minocycline is less common than to penicillin and cephalosporin, in which the symptoms develop
following oral administration rather than by topicaladministration as in minocycline hydrochloride ointment1,17）. Minocycline hydrochloride ointment is a den-tal ointment which was applied to the gingival sulcus

tal antibiotic formulation that is applied locally to peri-

for treating periodontitis.

odontal pockets, usually once a week, for the treatment

We conducted an open test for possible allergy to allof periodontitis. Minocycline hydrochloride dental oint-

the materials used for her dental treatment on thement 2％（Periofeel Dental Ointment 2％, Showa

inner side of the patient’s upper arm. The results wereYakuhin Kako Corp.）（1 syringe 0.5 g）contains 10 mg

all negative after 15 minutes, 30 minutes and 60 min-of minocycline hydrochloride. As of October 2022, theutes. A subsequent prick test showed a positive reac-

Pharmaceuticals and Medical Devices Agency（PMDA）

tion to minocycline hydrochloride dental ointment.

has reported 12 cases of adverse reactions to minocy-

Eventually, acidulated phosphate fluoride jelly showed acline hydrochloride ointment in Japan（Table 2）. Mino-negative reaction（Fig. 2）. The family dentist and thecycline hydrochloride ointment is contraindicated inpatient were informed about the positive allergic reac-patients with a history of hypersensitivity to the tetra-tion to minocycline hydrochloride. The dentist was

cycline group of antibiotics, and the risk of anaphylaxis

advised to discontinue the usage of minocycline hydro-or urticaria is naturally high when used in patients withchloride dental ointment for this patient.

any allergies to internal medicines. In the reported
cases（Table 2）, it is not clear whether there was a

Clinical Course

history of allergy to antibiotics in the past, and it is also
not clear whether the onset occurred after multiple

For the next six months, she received maintenanceusage. In this case, there was no history of any allergic

treatment by the family dentist on three occasions, but

symptoms to antibiotics in the past, and there was nono subsequent allergic symptoms were observed.

history of long-term administration of antibiotics suchas minocycline hydrochloride（Minomycin, Pfizer Inc.,

Discussion

New York, USA）
. A sleep-inducing drug, Zolpidem Tartrate（Myslee Tablets, Astellas Pharma Inc., Tokyo,

Drug allergy is an immune-mediated reaction. Most

Japan）, was prescribed for insomnia by a family physi-

medicines have a small molecular weight, so antigenic-

cian. She had no other significant medical history.

ity is low. However, they may become antigenic byMoreover, no allergic symptoms developed in February

binding with macromolecules such as protein in vivo.

and March 2017 when periodontal treatment was per-

Antimicrobial and antifebrile agents are particularly

formed without using minocycline hydrochloride oint-ODEP Vol. 3, No. 1

156
Table 2No.

Year ofoccurrenceSide effect case list

Sex

Age

Symptoms

Presence of suspected concomitant medication

1

2005

Female

60’s

Vomiting, dyspnea, rash, erythema,pruritus

―

2

2006

Male

40’s

Anaphylactic shock

―

3

2006

Male

50’s

4

2009

Male

60’s

Shock symptoms

5

2010

Male

60’s

Laryngeal edema, whole body erythema

―

6

2014

Male

50’s

Anaphylactoid reaction

―

7

2014

Female60’s

Drug eruption

Mecobalamin, Adenosine 5’-Triphosphate Disodium Hydrate, Isosorbide, Pitavastatin Calcium

8

2015

Female20’s

Shock symptoms

―

9

2018

Male

70’s

Decreased level of consciousness, rash

―

10

2018

Male

70’s

Anaphylactic shock

Pranoprofen, Teprenone, Amoxicillin Hydrate,
Ethyl Aminobenzoate, Benzethonium Chloride,
Lidocaine Hydrochloride/Adrenaline

11

2019

Female60’s

Hypersensitivity

―

12

2019

Female50’s

Anaphylactic shock―

Anaphylactic shock（details unknown） Cefteream Pivoxil, Loxoprofen Sodium Hydrate,
Acrinol Hydrate
Hydrogen Peroxide

Twelve cases of side effects of minocycline hydrochloride ointment were reported to the Pharmaceuticals Medical Devices
Agency of Japan.

ment. Although a prick test for minocycline hydrochlo-ointment is used over an extended period of time15）.

ride was not conducted, it is possible that the additivein the dental ointment was the cause. However, current

Conclusion

reports only suggest the possibility of diarrhea fromhigh intake of such additives, and the likelihood of caus-

An incremental application of a small amount of topi-

ing urticaria, as observed in this case, is considered tocal dental antibiotics into the periodontal pocket canbe low. Additionally, while the additive hypromellose isresult in an immediate-type allergic reaction. It is

used in drug capsules, the patient had no recollection ofimportant to document such cases so that dental practi-

an allergy to capsule-shaped medications. This suggests

tioners can be aware of documented allergies to variousthat the minocycline hydrochloride ointment used in thedental materials for a proper diagnosis and to avoid

dental clinic resulted in sensitization/onset, causing her

such allergic reactions in the clinical practice.

to develop allergic symptoms after her periodontal
treatment in June. Minocycline hydrochloride ointment

Acknowledgements

contains 10 mg of minocycline hydrochloride and, evenWe thank Helen Jeays, BDSc AE, from Edanz Group（www.edanzediting.com/ac）for editing a draft of this manuscript.

if all the syringe is used, it is less than 1/10 of the doseof oral minocycline（100‒200 mg）. However, when it isused for periodontal maintenance, the duration of use isoften long. Knowles et al. suggested that taking oral

Conflict of Interest

minocycline for more than 2 years increases the likeli-

Conflict of Interest and Source of Funding:This study was partly supported by“The initiative forRealizing Diversity in the Research Environment（Distinc-hood of allergies. We recommend that these risk factors
be taken into account when minocycline hydrochlorideDec, 2023

Hypersensitivity to Dental Antibiotics

157

tive Features Type and Collaboration Type）”of the Japa-

7）Haskes C, Shea M, Imondi D. Minocycline-induced

nese Ministry of Education, Culture, Sports, Science andTechnology and Grant-in-Aid for Scientific Research ofscleral and dermal hyperpigmentation. Optom Vis Sci

Japan（20K10038）. The authors state explicitly that there
are no conflicts of interest in connection with this article.

8）Hung PH, Caldwell JB, James WD. Minocycline-induced
hyperpigmentation. J Fam Pract 1995; 41: 183‒185.
9）Krause W. Drug-induced hperpigemntation: a systematic
review. J Dtsch Dermatol Ges 2013; 11: 644‒651.
10）Siller GM, Tod MA, Savage NW. Minocycline-induced
oral pigmentation. J Am Acad Dermatol 1994; 30: 350‒
354.
11）Treister NS, Magalnick D, Woo SB. Oral mucosal pigmentation secondary to minocycline therapy: report oftwo cases and a review of the literature. Oral Surg Oral
Med Oral Pathol Oral Radiol Endod 2004; 97: 718‒725.
12）Okano M, Imai S. Anaphylactoid symptoms due to oral
minocycline. Acta Derm Venereol 1996; 76: 164.
13）Hitchcock MG, Williford PM, Clayton BD. Minocycline
hypersensitivity with respiratory failure. Arch Dermatol 1999; 135: 139‒140.
14）Jang JW, Bae YJ, Kim YG, Jin YJ, Park KS, Cho YS,
Moon HB, Kim TB. A case of anaphylaxis to oral minocycline. J Korean Med Sci 2010; 25: 1231‒1233.
15）Knowles SR, Shapiro L, Shear NH. Serious adverse reactions induced by minocycline. Report of 13 patients andreview of the literature. Arch Dermatol 1996; 132: 934‒
939.
16）Hamilton LA, Guarascio AJ. Tetracycline allergy. Pharmacy（Basel）2019; 7: 104.
17）Baldo BA, Pham NH. Drug Allergy: Clinical Aspects,
Diagnosis, Mechanisms, Structure-Activity Relationships. Springer: Berlin: 2013. 1‒181, 245‒313.

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ODEP Vol. 3, No. 1

158

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13．The copyrights for articles published in ODEP belong to the Japanese Society of Conservative Dentistry.
14．Matters not mentioned in these guidelines will be independently determined by the editorial board.
Submission of your manuscript for publication must conform to the following“Submission Guidance”as well as“Submission Guidelines.”Dec, 2023

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The publishing charge is 10,000 yen for a Journal page including tables and figures. Extra charges for such as
figures and tables preparation, color printing of photographs will also be paid by the authors. If authors do not
pay publishing charge, the article may be retracted.

Submission Guidance（applied as of the Issue 1 of Vol. 4）
Manuscript organization
1．In principle, original articles should be organized into the following sections:（1）cover page;（2）abstract;（3）Main text（Introduction, Materials and Methods, Results, Discussion, and Conclusion）;（4）References; and（5）Figure and table captions. Page numbering should start with the cover page. In principle, manuscripts, such as
reviews or case reports, other than original articles should be organized in the same format as that of originalarticles.
2．Manuscript sections
1） Title: The title should concisely describe the contents of the manuscript. The subtitle should also clearly
describe the contents, and should not consist of only numbers.
2） Introduction: The introduction should clearly describe the background, novelty, purpose, and significance ofthe study.
3） Materials and Methods: This section should provide detailed information on the materials, equipment, or
methods used along with clear instructions so that the experiments can be reproduced by others. Parameter settings, number of specimens, extraction methods, statistics processing, and others should comply withthe purpose of the study.
4） Results: This section should simply present the findings without bias or interpretation. Measurement results
should show characteristic values including mean values and standard deviations.
5） Discussion: This section should carefully consider the materials and methods, results, and others referring
to relevant literature. Please note that it should not be overly assertive and should avoid off-topic points.
The discussion should stay focused on the study purpose and not digress into a general discussion.
6） Conclusion: The section should precisely summarize the results obtained and relate them to the purpose of
the study and hypothesis as presented in the Introduction.
3．Manuscripts should be prepared using A4-size paper. The suggested length of each typed page is 80 alphanumeric characters per line×25 lines per page using a 12-point font. Top/bottom/left/right margins should beapproximately 25 mm. Non-Japanese names and places should be in their original names.
4．For the manuscript style, refer to the latest issue of the journal.
Ethics code
1．Each report on the result of clinical research（clinical trial or observational research）or research involving any
specimen collected from a human body must include a clear statement that it was approved by the head of theaffiliated institution or by the research ethics review board assigned by the institution’s head, in order toexpressly indicate that the research was conducted in compliance with all applicable guidelines and laws,
including the Declaration of Helsinki and the medical research guidelines, etc. issued by the Ministry of Health,Labour and Welfare including the following:
1）Ethical Guidelines for Life Science and Medical Research Involving Human Subjects;
2）Guidelines for Gene Therapy and Other Clinical Research; and3）Clinical Trials Act.
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in the Act on the Safety of Regenerative Medicine must include a clear statement that the technology was provided to the patients in compliance with the aforementioned Act.
3．Each report on a case of a therapeutic method involving any off-label drug or device, or any pharmaceutical,
medical device, regenerative medicine, or other related product not domestically approved must include a clear
statement that the use was approved by the committee concerned（research ethics review board, review
board for unapproved new drugs, etc.）
at the affiliated institution or that the case report was approved for publication by ODEP’s Clinical and Epidemiological Ethics Committee.
4．In each instance of publication of an academic paper, all personal information must be thoroughly protected sothat none of the research subjects（patients）can be identified from it.
5．In each instance where a patient’s clinical photo or X-ray image is included in an academic paper for publication,a clear statement must be provided that the consent of each such patient（or a parent, guardian, or proxy if thepatient is a minor or in case it is otherwise difficult to obtain consent from the patient）was duly obtained.
6．Each report on the result of research involving animal subjects must include a clear statement that the research
was approved by the animal experiment committee, etc. at the affiliated institution.
Cover page
1．The title, authors’ names, institutional affiliations, and corresponding author’s contact address should be centered,with a new line for each item, on the cover page.
2．The title should be in upper and lowercase letters, where the first letter of each word is uppercased and theremaining letters are lowercased. Articles, prepositions, conjunctions, and commonly used technical terms arelowercased. For hyphenated compound words, the letters following the hyphen should be lowercased.
3．The corresponding author’s contact details should include the following information: one author’s name, institutional affiliation, postal address, telephone and fax numbers, and e-mail address.
Abstract
1．The abstract should be a maximum of 400 words organized into four sections with the following headings: Purpose, Methods, Results, and Conclusion. Approximately three keywords should be placed at the end of theabstract.
2．Contributors should put considerable effort into preparing the abstract as it may determine whether or not thereader continues with the manuscript. When necessary, abstracts should be checked by a native English
reviewer（preferably with expertise in dental medicine）.
Main text
1．Introduction, Materials and Methods, Results, Discussion, and Conclusion are the main headings and are notnumbered.
2．Subparagraphs should be numbered in the following order: 1. 2. 3. …; 1）2）3）…;（1）（2）（3）…; ① ② ③ …;
and a. b. c. …
3．English characters should be written in the following manner:
1） Generally, only the last name should be used to indicate a person.
2） When the name of a product or manufacturer must be in the original language, the first letter of each word
should be uppercased and the remaining letters lowercased.
In principle,“generic name（product name, company name, city［state in the case of U.S.］, country）”
shouldbe used in English manuscripts. Trademark and registration symbols ® and TM are not required.
3） Regarding common nouns in German or Latin, the first letter should be uppercased and the remaining letters lowercased. For common nouns in English and French, all letters should be lowercased.
4） Regarding binominal nomenclature, the first letter of the genus should be uppercased and the remaining
letters lowercased. The names of all genera and species should be italicized. When the same genusDec, 2023

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appears frequently, it is acceptable to replace the name with the initial after the first use.
Example: Streptococcus mutans → S. mutans
5） For nouns that must be in their original language, other than German, Latin, English or French, all lettersshould be lowercased, except for commonly used technical terms.
4．In principle, SI units are used for measurements.
5．Any conflicts of interests（COI）must be declared after the conclusions. When there is no COI, the statement“The authors declare no conflict of interest related to this paper”should be included.
6．Acknowledgments for all sources supporting this study including grant funds should be added after of COI.
References
1．References must be listed at the end of the main text, and numbered in the same order as they appear in thetext.
2．In the main text, a cited reference should appear with a superscript numeral and closing parenthesis. When two
references are cited, a comma should be used to separate them; more than two references should be connectedby an en dash between the first and last numeral.
Examples:“by authors3）”,“…is reported7,8）”,“previous studies10‒15）show”
3．Examples of Reference
a ．Journal articles
Number）Last name and first name of all authors with a comma separating each author. Title of paper. Name ofjournal and publishing year in the Christian era; Volume number: Inclusive page numbers of paper.
Example:1）Clark AB, Erickson D, Hamilton FG. Tensile bond strength and modulus of elasticity of several composite resins. J Dent
Res 1992; 37: 618‒621.

b ．Book
Number）Author（co-authors）. Title of book. First/last volume. Edition. Publisher’s name: Publisher’s location（City）; Publishing year in the Christian era. Cited pages.
Example:2）Phillips RW. Skinner’s science of dental materials. 9th ed. WB Saunders: Philadelphia; 1991. 219‒221.

c ．Book with co-authors
Number）Contributor’s name. Title of contributed article. Name of editor（editor-in-chief）. Book title. First/lastvolume. Edition. Publisher’s name: Publisher’s location（City）; Publishing year in the Christian era.
Cited pages.
Example:3）Torneck CD. Dentin-pulp complex. Ten Cate AR. Oral histology. 5th ed. Mosby: St. Louis; 1998. 150‒196.

For cases in which each author’s contribution is not separately indicated, the author’s name and the title of thecontributed article should not be listed.
Number）Name of editor（editor-in-chief）. Title of book. First/last volume. Edition. Publisher’s name: Publisher’slocation（City）; Publishing year in the Christian era. Cited pages.
d ．Other writing styles
・Abstracts of scientific meeting
Number）Presenter（all presenters should be cited. A comma should be used to separate the authors in the caseof co-presentations.）. Title of abstract. Name of journal and publishing year in the Christian era; Volume number: Inclusive page numbers, abstract number.
Example:4）Marais JT. Cleaning efficacy of a new root canal irrigation material. J Dent Res 1998; 77: 669, Abst. No. 300.

・ Journal articles in press
In principle, the same style as that of a regular journal article should be used; however, when the inclusive page

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numbers are not known, they can be omitted. The statement“in press”should be shown at the end.
Example:5）Sato K. Effect of toothbrushes on gingival abrasion. J Periodont Res 1994; 29: in press.

・Electronic journal
In principle, the same style as that of a regular journal article should be used; however, when the inclusive page
numbers are not known, the DOI and other indicators should be shown. When papers are published in electronic journals before printing, the statement［Epub ahead of print］should be shown after the publishing year
and month.
Example:6）Sunada N, Ishii R, Shiratsuchi K, Shimizu Y, Tsubota K, Kurokawa H, Miyazaki M. Ultrasonic measurement of theeffects of adhesive application and power density on the polymerization behavior of core build-up resins. Acta Odontol Scand; doi: 10.3109/00016357.2011.654252

・Internet website
Page publisher. Title of page. URL address.（Access date）
Example:7）World Health Organization. Continuous improvement of oral health in the 21st century. http://www.who.int/oral_
health/en/（cited 2005. 10. 1）

4．In principle, journal names should be abbreviated in accordance with the format used by the journal.
Figures and tables
1．Figures, photographs, and tables are categorized into figures and tables, and then numbered. Paper size shouldbe A4, and each figure and table should be printed on a separate page. The numbers allocated for the figures
and tables should be consistent with those referred to in the text.
2．Figures and tables should be accompanied by explanations that are easily understandable. Explanations of figures/tables are presented as captions and explanations for tables/tables should be presented as footnotes.
3．If authors wish to have the figures shown in color, color data should be attached; if authors wish to have thefigures shown in black and white, black-and-white data should be attached.
Notes on creating imaging data:
・ The jpg data format should be used if possible.
・ Image size should correspond to the layout; image resolution should be at least 300 dpi for photographs andat least 1,200 dpi for line drawings.
4．If authors wish to reprint a figure from another publication, should be responsible for confirming whether permission is required from that publication, and obtaining permission if necessary. In addition, the source should beclearly stated in the explanatory text of each figure.
Sending manuscript intended for publication
1．The manuscript（cover page, abstract, main text, references, and figure and table captions are created as onefile）should be formatted as a Microsoft Office Word（hereafter“Word”）document.
2．Figures should be provided in jpg or pdf format.
3．Tables should be provided in Microsoft Office Excel, jpg, pdf, or Word format.
4．File titles should be as follows:“author name”_“university name（name after department is not needed）”_“manuscript/figure/table/submission form”_“.filename extension（indicating file type）”.
Example）Nihon Tarou_Nihon University/manuscript.docx; Nihon Taro_Nihon University_Figure.jpg; Nihon
Taro_Nihon Univesity_Table.xlsx; Nihon Taro_Nihon University_submission form.pdf
Also, pdf files with embedded fonts for all contents are acceptable for submission. In such cases, file names
should be as follows: Nihon Taro_Nihon University_comprehensive manuscript.pdf
5．E-mail title（subject）should be“Submitted papers for ODEP”.

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6．Submitted papers should be sent to the e-mail address of Oral Health Association of Japan: hensyu6@kokuhoken.
or.jp. For safety, also send as a CC to hensyu5@kokuhoken.or.jp.
7．When e-mail submission is difficult for such reasons as the file size is too large, submission via an FTP server
and other methods are acceptable. In such cases, the intent of submission should be informed by e-mail. In thee-mail, information regarding the site and other methods for downloading the file should be provided.
8．The submission form of ODEP provided on the Japanese Society of Conservative Dentistry’s home page（http://www.kokuhoken.or.jp/exterior/ jscd/fileform/）can be used for submission.

Reprographic Reproduction
The Japanese Society of Conservative Dentistry authorized Japan Academic Association For Copyright
Clearance（JAC）to license our reproduction rights of copyrighted works. If you wish to obtain permissionsof these rights, please refer to the homepage of JAC（http://www.jaacc.org/en/）and confirm appropriateorganizations to request permission.

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